Immunogenic wt-1 peptides and methods of use thereof

ABSTRACT

This invention provides peptides, immunogenic compositions and vaccines, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising heteroclitic peptides derived from the WT-1 protein.

GOVERNMENT SUPPORT

This application was supported by grant P01 23766 from the NationalInstitutes of Health. The government has rights in this invention.

FIELD OF INVENTION

This invention provides peptides, compositions and vaccines comprisingsame, and methods of treating, reducing the incidence of, and inducingimmune responses to a WT1-expressing cancer, comprising administeringsame.

BACKGROUND OF THE INVENTION

Wilms tumor (WT), a pediatric nephroblastoma that occurs with afrequency of 1 in 10,000 births, has been the subject of intenseclinical and basic research for several years. The tumor is embryonic inorigin, it is detected in children usually during the first 5 years oflife and can occur unilaterally or bilaterally. A WT arises whencondensed metanephric mesenchymal cells of the developing kidney fail toproperly differentiate. The implication of the Wilms tumor 1 (WT1) tumorsuppressor gene in the etiology of WT illustrated the impact thatgenetic alterations can have on both development and tumorigenesis.

Wilms tumor protein I (WT1) is a zinc finger transcription factorexpressed during normal ontogenesis such as in fetal kidney, testis andovary. In adults, WTI expression is limited to low levels onhematopoietic stem cells, myoepithelial progenitor cells, renalpodocytes and some cells in testis and ovary. Recent demonstration thatWTI is over expressed in several types of leukemia suggested that WTIwould be an attractive target for immunotherapy for various cancers.

The Wilms' tumor oncogene protein (WT1) is an attractive target forimmunotherapy for leukemias and a wide range of cancers. Peptidesderived from the WT1 protein have been identified that induceHLA-A0201-restricted cytotoxic CD8 T cells, capable of killing tumorcells. Two peptides that bind to HLA-A0201 (RMFPNAPYL; SEQ ID NO:56) orHLA-A2402 (CMTWNQMNL; SEQ ID NO:57) have been extensively studiedworldwide and have been in clinical trials in patients with leukemia andother solid tumors (Oka et al., Scientific World Journal 2007; 7:649-665; Mundlos et al. Development 1993; 119:1329-41; Keilholz et al.Leukemia 2005; 19: 1318-1323). These results are encouraging and haveprovided strong evidence and a rational for therapeutic targeting of theWT1-derived T cell epitopes for leukemias and a wide range of humancancers.

The therapeutic application of the above two WT1-derived peptides islimited to the people who are HLA-A0201, an HLA haplotype found in about40% of Caucasians and HLA-A2402, a found haplotype in about 40% ofJapanese and other Asian populations. Therefore, there is an unmet needfor WT1-derived peptides that might be used for most of the world'spopulations. To extend the therapeutic application in a broader range ofpopulation, novel peptides derived from WT1 protein that bind tomultiple HLA haplotypes are desired. Such peptides would therefore becapable of stimulating T cells from a larger percentage of the targetpopulation, allowing a vaccine strategy that would address a largesegment of the population, with durable cytotoxic memory cells.

SUMMARY OF THE INVENTION

This invention provides peptides, compositions, and immunogeniccompositions such as vaccines comprising immunogenic peptides, andmethods of treating, reducing the incidence of, and inducing immuneresponses to a WT1-expressing cancer, comprising administeringimmunogenic peptides, or stimulating T cells outside of a human patientthat can then be infused into the patient for treatment.

In one embodiment, the present invention provides an isolated peptidehaving an amino acid (AA) sequence consisting of any one of thesequences SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46,47, 48, 49, 50 and 55. In one embodiment, the present invention providesan isolated HLA class I binding peptide having an amino acid (AA)sequence consisting of any one of the sequences SEQ ID NO:6, 7, 30, 31,32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48. In one embodiment, thepresent invention provides an isolated HLA class II binding WT1 peptidehaving an amino acid (AA) sequence consisting of any one of thesequences SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 39, 43, 44, 46, 49, 50 and 55.

In one embodiment, the present invention provides an isolated peptidehaving an amino acid (AA) sequence consisting of any one of thesequences SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46,47, 48, 49, 50 and 55, or a fragment of any one of the foregoing. In oneembodiment, the present invention provides an isolated HLA class Ibinding peptide having an amino acid (AA) sequence consisting of any oneof the sequences SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41,42, 47 and 48 or a fragment of any one of the foregoing. In oneembodiment, the present invention provides an isolated HLA class IIbinding WT1 peptide having an amino acid (AA) sequence consisting of anyone of the sequences SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55 or a fragment ofany one of the foregoing.

In another embodiment, the present invention provides a compositioncomprising (a) an antigen-presenting cell and (b) a peptide selectedfrom SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47,48, 49, 50 and 55. In another embodiment, the present invention providesa composition comprising (a) an antigen-presenting cell and (b) an HLAclass I binding peptide selected from SEQ ID NO: 6, 7, 30, 31, 32, 33,34, 35, 36, 37, 38, 41, 42, 47 and 48. In another embodiment, thepresent invention provides a composition comprising (a) anantigen-presenting cell and (b) an HLA class II binding peptide selectedfrom SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 39, 43, 44, 46, 49, 50 and 55.

In another embodiment, the present invention provides a vaccinecomprising one or more peptides of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37,38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In anotherembodiment, the present invention provides a vaccine comprising one ormore HLA class I binding peptides selected from SEQ ID NO: 6, 7, 30, 31,32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48. In another embodiment,the present invention provides a vaccine comprising one or more HLAclass II binding peptides selected from SEQ ID N08, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55.In another embodiment, the present invention provides a vaccinecomprising one or more HLA class I binding peptides selected from SEQ IDNO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50and 55, and one or more HLA class II binding peptides selected from SEQID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39,43, 44, 46, 49, 50 and 55.

In another embodiment, the present invention provides a method oftreating a subject with a WT1-expressing cancer, the method comprisingadministering to the subject a peptide or vaccine of the presentinvention, thereby treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a peptide orvaccine of the present invention, thereby reducing the incidence of aWT1-expressing cancer, or its relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing an anti-cancer immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising (a) a WT1 protein; (b) a modified fragment of aWT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) anucleotide molecule encoding a modified fragment of a WT1 protein,thereby inducing an anti-mesothelioma immune response in a subject. Inone embodiment, the modified fragment of a WT1 protein consists of apeptide or comprises a peptide from among SEQ ID NO: 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36,37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55.

In another embodiment, the present invention provides a method oftreating a subject with a cancer, the method comprising the step ofadministering to the subject an immunogenic composition comprising (a) aWT1 protein; (b) a modified fragment of a WT protein; (c) a nucleotidemolecule encoding a WT1 protein; or (d) a nucleotide molecule encoding amodified fragment of a WT1 protein, thereby treating a subject with amesothelioma. In one embodiment, the modified fragment of a WT1 proteinis a peptide from among SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41,42, 43, 44, 46, 47, 48, 49, 50 and 55.

In another embodiment, the present invention provides a method ofreducing an incidence of a cancer, or its relapse, in a subject, themethod comprising the step of administering to the subject animmunogenic composition comprising (a) a WT1 protein; (b) a modifiedfragment of a WT protein; (c) a nucleotide molecule encoding a WT1protein; or (d) a nucleotide molecule encoding a modified fragment of aWT1 protein, thereby reducing an incidence of a mesothelioma, or itsrelapse, in a subject. In one embodiment, the fragment of a WT1 proteinis a peptide from among SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41,42, 43, 44, 46, 47, 48, 49, 50 and 55.

In another embodiment, the cancer is a WT1-expressing cancer. In oneembodiment, the WT1-expressing cancer is an acute myelogenous leukemia(AML). In another embodiment, the WT1-expressing cancer is associatedwith a myelodysplastic syndrome (MDS). In another embodiment, theWT1-expressing cancer is an MDS. In another embodiment, theWT1-expressing cancer is a non-small cell lung cancer (NSCLC). Inanother embodiment, the WT1-expressing cancer is a Wilms' tumor. Inanother embodiment, the WT1-expressing cancer is a leukemia. In anotherembodiment, the WT1-expressing cancer is a hematological cancer. Inanother embodiment, the WT1-expressing cancer is a lymphoma. In anotherembodiment, the WT1-expressing cancer is a desmoplastic small round celltumor. In another embodiment, the WT1-expressing cancer is amesothelioma. In another embodiment, the WT1-expressing cancer is amalignant mesothelioma. In another embodiment, the WT1-expressing canceris a gastric cancer. In another embodiment, the WT1-expressing cancer isa colon cancer. In another embodiment, the WT1-expressing cancer is alung cancer. In another embodiment, the WT1-expressing cancer is abreast cancer. In another embodiment, the WT1-expressing cancer is agerm cell tumor. In another embodiment, the WT1-expressing cancer is anovarian cancer. In another embodiment, the WT1-expressing cancer is auterine cancer. In another embodiment, the WT1-expressing cancer is athyroid cancer. In another embodiment, the WT1-expressing cancer is ahepatocellular carcinoma. In another embodiment, the WT1-expressingcancer is a thyroid cancer. In another embodiment, the WT1-expressingcancer is a liver cancer. In another embodiment, the WT1-expressingcancer is a renal cancer. In another embodiment, the WT1-expressingcancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressingcancer is a sarcoma. In another embodiment, the WT1-expressing cancer isany other carcinoma or sarcoma.

In another embodiment, the WT1-expressing cancer is a solid tumor. Inanother embodiment, the solid tumor is associated with a WT1-expressingcancer. In another embodiment, the solid tumor is associated with amyelodysplastic syndrome (MDS). In another embodiment, the solid tumoris associated with a non-small cell lung cancer (NSCLC). In anotherembodiment, the solid tumor is associated with a lung cancer. In anotherembodiment, the solid tumor is associated with a breast cancer. Inanother embodiment, the solid tumor is associated with a colorectalcancer. In another embodiment, the solid tumor is associated with aprostate cancer. In another embodiment, the solid tumor is associatedwith an ovarian cancer. In another embodiment, the solid tumor isassociated with a renal cancer. In another embodiment, the solid tumoris associated with a pancreatic cancer. In another embodiment, the solidtumor is associated with a brain cancer. In another embodiment, thesolid tumor is associated with a gastrointestinal cancer. In anotherembodiment, the solid tumor is associated with a skin cancer. In anotherembodiment, the solid tumor is associated with a melanoma.

In another embodiment, the present invention provides a compositioncomprising an isolated peptide of the invention in combination with atleast 1 additional peptide. In certain embodiments, a compositioncomprising at least 2 different isolated peptides of the presentinvention is provided. In certain embodiments, a composition comprisingat least 3 or at least 4 different isolated peptides of the presentinvention is provided. Each possibility represents a separate embodimentof the present invention. In certain embodiments, the composition of thepresent invention is a vaccine.

In another embodiment, the present invention provides a method oftreating a subject with a WT1-expressing cancer, the method comprisingadministering to the subject a peptide or composition of the presentinvention, thereby treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a peptide orcomposition of the present invention, thereby reducing the incidence ofa WT1-expressing cancer, or its relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of a WT1 protein-specific CTL, themethod comprising contacting a lymphocyte population with a peptide orcomposition of the present invention, thereby inducing formation andproliferation of a WT1 protein-specific CTL. This method can beconducted in vitro, ex vivo or in vivo. When conducted in vitro or exvivo, these CTL can then be infused into a patient for therapeuticeffect.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of (a) a WT1 protein-specific CD8⁺lymphocyte; or (b) a CD4⁺ lymphocyte specific for the WT1 protein, orthe combination thereof, the method comprising contacting a lymphocytepopulation with a peptide or composition of the present invention,thereby inducing formation and proliferation of (a) a WT1protein-specific CD8⁺ lymphocyte; or (b) a CD4⁺ lymphocyte specific forthe WT1 protein; or a combination thereof. This method can be conductedin vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, theseCTL can then be infused into a patient for therapeutic effect.

BRIEF DESCRIPTION OF THE FIGURES

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention are briefly summarized. Details of the above may be hadby reference to certain embodiments thereof, which are illustrated inthe appended drawings. These drawings form a part of the specification.It is to be noted; however, that the appended drawings illustratepreferred embodiments of the invention and therefore are not to beconsidered limiting in their scope. In the figures herein, the set ofclustered data bars in the graphs for each peptide are presented in thesame order from left to right as in shown the figure legend from top tobottom.

FIG. 1 shows the results of a T2 stabilization assay showing thatbinding of NLMNLGATL peptide to HLA-A2 molecule is stronger thanNQMNLGATL and NYMNLGATL peptides. Native NQMNLGATL, heterocliticNLMNLGATL or NYMNLGATL was pulsed onto T2 cells at the indicatedconcentrations as described in the Materials and methods. Thestabilization of the HLA-A2 molecule by the peptides was measured by theexpression of HLA-A2 molecule;

FIG. 2 A-B show that NLMNLGATL peptide induces strong peptide-specific Tcell response which cross-reacts to its native sequence NQMNLGATL. Foreach peptide, the bars represent, from left to right, CD14, Nativepeptide, NLMNLGATL and PSMA, respectively. CD3 T cells from a healthyHLA-A0201 homozygous donor was stimulated with either NQM or NLMNLGATLpeptide for 3 (A) or 5 (B) rounds. The peptide-specific response wasmeasured by the IFN-g secretion upon challenged with individual peptide.Each data point represents average+/−SD from triplicate cultures;

FIG. 3 shows that NLMNLGATL peptide induces cytotoxicity of T cellsagainst WT1+HLA-A0201+ leukemia cells. The T cells from an HLA-A0201positive donor were stimulated with NLMNLGATL peptide for 5 rounds. Thecytotoxicity of the cells were measured by 5 hr-⁵¹Cr release assayagainst AML cell line SET-2 (WT1+, HLA-A0201+), HL-60 (WT1+, HLA-A0201−)or primary leukemia blasts from a HLA-A2 positive patient. Each datapoint represents average+/−SD from triplicate cultures;

FIG. 4 A-B show peptide-specific T cell response in HLA-A2402 donor. CD3T cells from a healthy HLA-A2402 homozygous donor was stimulated withNQMNLGATL, NLMNLGATL or NYMNLGATL peptides for 3 (A) or 5 (B) rounds.For each peptide, the bars represent, from left to right, CD14, Nativepeptide, NLMNLGATL, NYMNLGATL and irrelevant peptide, respectively. Thepeptide-specific response was measured by the IFN-g secretion uponchallenged with individual peptide. Each data point representsaverage+/−SD from triplicate cultures;

FIG. 5 A-B show HLA-DR.B1 peptide-specific T cell responses. (A). CD3 Tcells were stimulated with DR-het-1 or DR-het-2 peptide for 5 rounds andthe epitope-specific response was measured by IFN-g Elispot assay. Foreach peptide, the bars represent, from left to right, CD14, Nativepeptide, NLMNLGATL, WT1-CMT, PSMA, DR-Nat-1/Nat-2, DR-het-1/het-2,B2A2L, BA25 and HL-60, respectively. (B). CD3 T cells were stimulatedwith short peptides NQMNLGATL, NLMNLGATL and long peptides DR-native-1,DR-native-2, DR-het-1 or DR-het-2 peptide for 5 rounds and thecytotoxicity was measured by ⁵¹Cr-release assay, against Leukemia cellline BA-25 (HLA-A2+/A24+, WT1+) and the control HL-60 cells. Each datapoint represents average+/−SD from triplicate cultures;

FIG. 6 depicts CD3 T cells from a HLA-B0702-positive donor werestimulated with 2 sets of peptides (total five) for 5 times in vitro.The peptide-specific response was measured by IFN-gamma ELISPOT assay,against individual peptide; peptides tested from left to right are SEQID NOS:34, 37, 38, 30 and 31; and for each peptide, the bars represent,from left to right, responses to CD14, Native-1 peptide, Het-1 toNative-1, Het-2 to Native-1, Native-2, Het-1 to Native-2 and control,respectively; and

FIG. 7 depicts the results of an ELISPOT assay using donor SA (5stimulations) for the Het-1 (SEQ ID NO:6) and Het-2 (SEQ ID NO:7) A24peptide, in comparison to the native sequence (SEQ ID NO:5). For eachpeptide, the bars represent, from left to right, CD14, A24-nativepeptide, A24-het-1, A24-het-2, A24-235, and PSMA, respectively. Theheteroclitic peptides generate cross-reactive responses.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides immunogenic peptides, and compositions andvaccines comprising immunogenic peptides, and methods of treating,reducing the incidence of, and inducing immune responses to aWT1-expressing cancer, comprising administering one or more immunogenicpeptides.

This invention provides synthetic peptides and methods of treating,reducing the incidence of, and inducing immune responses against aWT1-expressing cancer, comprising immunogenic peptides.

The WT1 molecule from which the peptides of the present invention arederived has, in another embodiment, the sequence:

(SEQ ID NO: 51)   1SRQRPHPGAL RNPTACPLPH FPPSLPPTHS PTHPPRAGTA AQAPGPRRLL  51AAILDFLLLQ DPASTCVPEP ASQHTLRSGP GCLQQPEQQG VRDPGGIWAK 101LGAAEASAER LQGRRSRGAS GSEPQQMGSD VRDLNALLPA VPSLGGGGGC 151ALPVSGAAQW APVLDFAPPG ASAYGSLGGP APPPAPPPPP PPPPHSFIKQ 201EPSWGGAEPH EEQCLSAFTV HFSGQFTGTA GACRYGPFGP PPPSQASSGQ 251ARMFPNAPYL PSCLESQPAI RNQGYSTVTF DGTPSYGHTP SHHAAQFPNH 301SFKHEDPMGQ QGSLGEQQYS VPPPVYGCHT PTDSCTGSQA LLLRTPYSSD 351NLYQMTSQLE CMTWNQMNLG ATLKGVAAGS SSSVKWTEGQ SNHSTGYESD 401NHTTPILCGA QYRIHTHGVF RGIQDVRRVP GVAPTLVRSA SETSEKRPFM 451CAYPGCNKRY FKLSHLQMHS RKHTGEKPYQ CDFKDCERRF SRSDQLKRHQ 501RRHTGVKPFQ CKTCQRKFSR SDHLKTHTRT HTGKTSEKPF SCRWPSCQKK 551FARSDELVRH HNMHQRNMTK LQLAL.

The foregoing sequence of the WT-1 protein is that published by Gessleret al. (Gessler M, Poustka A, Cavenee W, Neve R L, Orkin S H, Bruns G A.Homozygous deletion in Wilms tumours of a zinc-finger gene identified bychromosome jumping. Nature. 1990; 343(6260):774-778. Prepublished on1990 Feb. 22 as DOI 10.1038/343774a0.) which comprises 575 amino acidsand includes the first 126 amino acids in the N-terminus missing in the(Exon 5+, KTS+) isoform of WT-116.

In another embodiment, the WT1 sequence is

MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL(GenBank Accession number AY245105; SEQ ID NO: 52).

In another embodiment, the WT1 molecule has the sequence:

AAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNM HQRNMTKLQLAL(GenBank Accession number NM_000378; SEQ ID NO: 53).

In another embodiment, the WT1 molecule has the sequence:

MQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHH NMHQRNMTKLQLAL(GenBank Accession number NP_077742; SEQ ID No: 54).

In another embodiment, the WT1 protein has the sequence set forth inGenBank Accession # NM_(—)024426. In other embodiments, the WT1 proteinhas or comprises one of the sequences set forth in one of the followingsequence entries: NM_(—)024425, NM_(—)024424, NM_(—)000378, 595530,D13624, D12496, D 12497, or X77549. In another embodiment, the WT1protein has any other WT1 sequence known in the art. This inventionprovides peptides, compositions, and immunogenic compositions such asvaccines comprising immunogenic peptides, and methods of treating,reducing the incidence of, and inducing immune responses to aWT1-expressing cancer, comprising administering immunogenic peptides. Insome cases, the peptides described herein are derived from peptides thatare native sequences of WT1, and may be referred to herein asWT1-derived peptides or as a WT1 peptide.

In one embodiment, the present invention provides an isolated WT1peptide having an amino acid (AA) sequence consisting of any one of thesequences SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46,47, 48, 49, 50 and 55. In one embodiment, the present invention providesan isolated HLA class I binding WT1 peptide having an amino acid (AA)sequence consisting of any one of the sequences SEQ ID NO:6, 7, 30, 31,32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48. In one embodiment, thepresent invention provides an isolated HLA class II binding WT1 peptidehaving an amino acid (AA) sequence consisting of any one of thesequences SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment the HLAclass I peptides consist of or comprise SEQ ID NO:6, 7, 30, 31, 32, 33,34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLA class II peptideconsists of or comprises SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55.

In one embodiment, the present invention provides an isolated WT1peptide having an amino acid (AA) sequence comprising any one of thesequences SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46,47, 48, 49, 50 and 55, or a fragment thereof. In one embodiment, thepresent invention provides an isolated HLA class I binding WT1-derivedpeptide having an amino acid (AA) sequence comprising of any one of thesequences SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47and 48. In one embodiment, the present invention provides an isolatedHLA class II binding WT1 peptide having an amino acid (AA) sequencecomprising of any one of the sequences SEQ ID NO:8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55.In another embodiment the HLA class I peptides consist of or compriseSEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48,and the HLA class II peptide consists of or comprises SEQ ID NO:8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46,49, 50 and 55.

In another embodiment, the present invention provides a compositioncomprising (a) an antigen-presenting cell and (b) a peptide selectedfrom SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47,48, 49, 50 and 55. In another embodiment, the present invention providesa composition comprising (a) an antigen-presenting cell and (b) an HLAclass I binding peptide selected from SEQ ID NO:6, 7, 30, 31, 32, 33,34, 35, 36, 37, 38, 41, 42, 47 and 48. In another embodiment, thepresent invention provides a composition comprising (a) anantigen-presenting cell and (b) an HLA class II binding peptide selectedfrom SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment the HLAclass I peptides consist of or comprise SEQ ID NO:6, 7, 30, 31, 32, 33,34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLA class II peptideconsists of or comprises SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55.

In another embodiment, the present invention provides a vaccinecomprising one or more peptides of SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38,39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment,the present invention provides a vaccine comprising one or more HLAclass I binding peptides selected from SEQ ID NO:6, 7, 30, 31, 32, 33,34, 35, 36, 37, 38, 41, 42, 47 and 48. In another embodiment, thepresent invention provides a vaccine comprising one or more HLA class IIbinding peptides selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. Inanother embodiment, the present invention provides a vaccine comprisingone or more HLA class I binding peptides selected from SEQ ID NO:6, 7,30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and one or moreHLA class II binding peptides selected from SEQ ID NO: 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and55. In another embodiment the HLA class I peptides consist of orcomprise SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47and 48, and the HLA class II peptide consists of or comprises SEQ IDNO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43,44, 46, 49, 50 and 55.

In another embodiment, the present invention provides a method oftreating a subject with a WT1-expressing cancer, the method comprisingadministering to the subject a WT1 peptide or vaccine of the presentinvention, thereby treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a WT1peptide or vaccine of the present invention, thereby reducing theincidence of a WT1-expressing cancer, or its relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing an anti-cancer immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising (a) a WT1 protein; (b) a fragment of a WTprotein; (c) a nucleotide molecule encoding a WT1 protein; or (d) anucleotide molecule encoding a fragment of a WT1 protein, therebyinducing an anti-mesothelioma immune response in a subject. In oneembodiment, the fragment of a WT1 protein consists of a peptide orcomprises a peptide from among SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39,41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment thefragment consists of a peptide or comprises a peptide from among SEQ IDNO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, or SEQID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39,43, 44, 46, 49, 50 and 55.

In another embodiment, the present invention provides a method oftreating a subject with a cancer, the method comprising the step ofadministering to the subject an immunogenic composition comprising (a) aWT1 protein; (b) a fragment of a WT protein; (c) a nucleotide moleculeencoding a WT1 protein; or (d) a nucleotide molecule encoding a fragmentof a WT1 protein, thereby treating a subject with a mesothelioma. In oneembodiment, the fragment of a WT1 protein is a peptide from among SEQ IDNO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50and 55. In another embodiment the fragment consists of a peptide orcomprises a peptide from among SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35,36, 37, 38, 41, 42, 47 and 48, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. Inanother embodiment the HLA class I peptides consist of or comprise SEQID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, andthe HLA class II peptide consists of or comprises SEQ ID NO:8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49,50 and 55.

In another embodiment, the present invention provides a method ofreducing an incidence of a cancer, or its relapse, in a subject, themethod comprising the step of administering to the subject animmunogenic composition comprising (a) a WT1 protein; (b) a fragment ofa WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) anucleotide molecule encoding a fragment of a WT1 protein, therebyreducing an incidence of a mesothelioma, or its relapse, in a subject.In one embodiment, the fragment of a WT1 protein is a peptide from amongSEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48,49, 50 and 55. In another embodiment the fragment consists of a peptideor comprises a peptide from among SEQ ID NO:6, 7, 30, 31, 32, 33, 34,35, 36, 37, 38, 41, 42, 47 and 48, or SEQ ID NO: 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55.In another embodiment the HLA class I peptides consist of or compriseSEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48,and the HLA class II peptide consists of or comprises SEQ ID NO:8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46,49, 50 and 55.

In another embodiment, the present invention provides a method oftreating a subject with a WT1-expressing cancer, the method comprisingadministering to the subject a WT1 peptide or vaccine of the presentinvention, thereby treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a WT1peptide or vaccine of the present invention, thereby reducing theincidence of a WT1-expressing cancer, or its relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing an anti-cancer immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising (a) a WT1 protein; (b) a fragment of a WTprotein; (c) a nucleotide molecule encoding a WT1 protein; or (d) anucleotide molecule encoding a fragment of a WT1 protein, therebyinducing an anti-mesothelioma immune response in a subject. In oneembodiment, the fragment of a WT1 protein is a peptide from among SEQ IDNO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50and 55. In another embodiment the fragment consists of a peptide orcomprises a peptide from among SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35,36, 37, 38, 41, 42, 47 and 48, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. Inanother embodiment the HLA class I peptides consist of or comprise SEQID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, andthe HLA class II peptide consists of or comprises SEQ ID NO:8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49,50 and 55.

In another embodiment, the present invention provides a method oftreating a subject with a cancer, the method comprising the step ofadministering to the subject an immunogenic composition comprising (a) aWT1 protein; (b) a fragment of a WT protein; (c) a nucleotide moleculeencoding a WT1 protein; or (d) a nucleotide molecule encoding a fragmentof a WT1 protein, thereby treating a subject with a mesothelioma. In oneembodiment, the fragment of a WT1 protein is a peptide from among SEQ IDNO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50and 55.

In another embodiment, the present invention provides a method ofreducing an incidence of a cancer, or its relapse, in a subject, themethod comprising the step of administering to the subject animmunogenic composition comprising (a) a WT1 protein; (b) a fragment ofa WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) anucleotide molecule encoding a fragment of a WT1 protein, therebyreducing an incidence of a mesothelioma, or its relapse, in a subject.In one embodiment, the fragment of a WT1 protein is a peptide from amongSEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48,49, 50 and 55.

In another embodiment, the cancer is a WT1-expressing cancer. In oneembodiment, the WT1-expressing cancer is an acute myelogenous leukemia(AML). In another embodiment, the WT1-expressing cancer is associatedwith a myelodysplastic syndrome (MDS). In another embodiment, theWT1-expressing cancer is an MDS. In another embodiment, theWT1-expressing cancer is a non-small cell lung cancer (NSCLC). Inanother embodiment, the WT1-expressing cancer is a Wilms' tumor. Inanother embodiment, the WT1-expressing cancer is a leukemia. In anotherembodiment, the WT1-expressing cancer is a hematological cancer. Inanother embodiment, the WT1-expressing cancer is a lymphoma. In anotherembodiment, the WT1-expressing cancer is a desmoplastic small round celltumor. In another embodiment, the WT1-expressing cancer is amesothelioma. In another embodiment, the WT1-expressing cancer is amalignant mesothelioma. In another embodiment, the WT1-expressing canceris a gastric cancer. In another embodiment, the WT1-expressing cancer isa colon cancer. In another embodiment, the WT1-expressing cancer is alung cancer. In another embodiment, the WT1-expressing cancer is abreast cancer. In another embodiment, the WT1-expressing cancer is agerm cell tumor. In another embodiment, the WT1-expressing cancer is anovarian cancer. In another embodiment, the WT1-expressing cancer is auterine cancer. In another embodiment, the WT1-expressing cancer is athyroid cancer. In another embodiment, the WT1-expressing cancer is ahepatocellular carcinoma. In another embodiment, the WT1-expressingcancer is a thyroid cancer. In another embodiment, the WT1-expressingcancer is a liver cancer. In another embodiment, the WT1-expressingcancer is a renal cancer. In another embodiment, the WT1-expressingcancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressingcancer is a sarcoma. In another embodiment, the WT1-expressing cancer isany other carcinoma or sarcoma.

In another embodiment, the WT1-expressing cancer is a solid tumor. Inanother embodiment, the solid tumor is associated with a WT1-expressingcancer. In another embodiment, the solid tumor is associated with amyelodysplastic syndrome (MDS). In another embodiment, the solid tumoris associated with a non-small cell lung cancer (NSCLC). In anotherembodiment, the solid tumor is associated with a lung cancer. In anotherembodiment, the solid tumor is associated with a breast cancer. Inanother embodiment, the solid tumor is associated with a colorectalcancer. In another embodiment, the solid tumor is associated with aprostate cancer. In another embodiment, the solid tumor is associatedwith an ovarian cancer. In another embodiment, the solid tumor isassociated with a renal cancer. In another embodiment, the solid tumoris associated with a pancreatic cancer. In another embodiment, the solidtumor is associated with a brain cancer. In another embodiment, thesolid tumor is associated with a gastrointestinal cancer. In anotherembodiment, the solid tumor is associated with a skin cancer. In anotherembodiment, the solid tumor is associated with a melanoma.

In another embodiment, the present invention provides a compositioncomprising an isolated peptide of the invention in combination with atleast 1 additional WT1-derived peptide. In certain embodiments, acomposition comprising at least 2 different isolated peptides of thepresent invention is provided. In certain embodiments, a compositioncomprising at least 3 or at least 4 different isolated peptides of thepresent invention is provided. Each possibility represents a separateembodiment of the present invention. In certain embodiments, thecomposition of the present invention is a vaccine.

In another embodiment, the present invention provides a method oftreating a subject with a WT1-expressing cancer, the method comprisingadministering to the subject a peptide or composition of the presentinvention, thereby treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT1-expressing cancer, or its relapse, in asubject, the method comprising administering to the subject a peptide orcomposition of the present invention, thereby reducing the incidence ofa WT1-expressing cancer, or its relapse, in a subject.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of a WT1 protein-specific CTL, themethod comprising contacting a lymphocyte population with a peptide orcomposition of the present invention, thereby inducing formation andproliferation of a WT1 protein-specific CTL. This method can beconducted in vitro, ex vivo or in vivo. When conducted in vitro or exvivo, these CTL can then be infused into a patient for therapeuticeffect.

In another embodiment, the present invention provides a method ofinducing formation and proliferation of (a) a WT1 protein-specific CD8⁺lymphocyte; or (b) a CD4⁺ lymphocyte specific for the WT1 protein, orthe combination thereof, the method comprising contacting a lymphocytepopulation with a peptide or composition of the present invention,thereby inducing formation and proliferation of (a) a WT1protein-specific CD8⁺ lymphocyte; or (b) a CD4⁺ lymphocyte specific forthe WT1 protein; or a combination thereof. This method can be conductedin vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, theseCTL can then be infused into a patient for therapeutic effect.

“Peptide,” in another embodiment of methods and compositions of thepresent invention, refers to a compound of subunit AA connected bypeptide bonds. In another embodiment, the peptide comprises an AAanalogue. In another embodiment, the peptide comprises a peptidomimetic.The different AA analogues and peptidomimetics that can be included inthe peptides of methods and compositions of the present invention areenumerated hereinbelow. The subunits are, in another embodiment, linkedby peptide bonds. In another embodiment, the subunit is linked byanother type of bond, e.g. ester, ether, etc. Each possibilityrepresents a separate embodiment of the present invention.

The unaltered peptides of the present invention (as described both aboveand below) are referred to collectively herein as “WT1 peptides.” Eachof the embodiments enumerated below for “WT1 peptides” applies tounaltered WT1 peptides and HLA class I and class II heterocliticpeptides of the present invention. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a WT1 peptide of the present invention binds toan HLA class I molecule or a class II molecule. In another embodimentthe peptide binds to both a class I and a class II molecule. In anotherembodiment, the HLA class II molecule is an HLA-DRB molecule. In anotherembodiment, the HLA class II-molecule is an HLA-DRA molecule. In anotherembodiment, the HLA molecule is an HLA-DQA1 molecule. In anotherembodiment, the HLA molecule is an HLA-DQB1 molecule. In anotherembodiment, the HLA molecule is an HLA-DPA1 molecule. In anotherembodiment, the HLA molecule is an HLA-DPB 1 molecule. In anotherembodiment, the HLA molecule is an HLA-DMA molecule. In anotherembodiment, the HLA molecule is an HLA-DMB molecule. In anotherembodiment, the HLA molecule is an HLA-DOA molecule. In anotherembodiment, the HLA molecule is an HLA-DOB molecule. In anotherembodiment, the HLA molecule is any other HLA class II-molecule known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the HLA class I molecule whose binding motif iscontained in or comprising a peptide of the present invention is, inanother embodiment, an HLA-A molecule. In another embodiment, the HLAclass I molecule is an HLA-B molecule. In another embodiment, the HLAclass I molecule is an HLA-C molecule. In another embodiment, the HLAclass I molecule is an HLA-A0201 molecule. In another embodiment, themolecule is HLA A1. In another embodiment, the HLA class I molecule isHLA A2. In another embodiment, the HLA class I molecule is HLA A2.1. Inanother embodiment, the HLA class I molecule is HLA A3. In anotherembodiment, the HLA class I molecule is HLA A3.2. In another embodiment,the HLA class I molecule is HLA A11. In another embodiment, the HLAclass I molecule is HLA A24. In another embodiment, the HLA class Imolecule is HLA B7. In another embodiment, the HLA class I molecule isHLA B27. In another embodiment, the HLA class I molecule is HLA B8. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the HLA class I molecule-binding WT1-derivedpeptide of methods and compositions of the present invention binds to asuperfamily of HLA class I molecules. In another embodiment, thesuperfamily is the A2 superfamily. In another embodiment, thesuperfamily is the A3 superfamily. In another embodiment, thesuperfamily is the A24 superfamily. In another embodiment, thesuperfamily is the B7 superfamily. In another embodiment, thesuperfamily is the B27 superfamily. In another embodiment, thesuperfamily is the B44 superfamily. In another embodiment, thesuperfamily is the C1 superfamily. In another embodiment, thesuperfamily is the C4 superfamily. In another embodiment, thesuperfamily is any other superfamily known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the HLA molecule is a A0101, A0201, A0203, A2402,A6901, B0702, A3101, B3501, B3503, B3508, B3802, B3801, B3901, B4001,B4402, B4701, B5701, C0401, C1701, DRB₁0101, DRB₁0402, DRB₁0402,DRB₁0401 or DRB₁1104 molecule. In another embodiment, the peptides ofSEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48,and SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 39, 43, 44, 46, 49, 50 and 55, bind to the HLA class I or class IImolecules described for each peptide in the Tables below. In anotherembodiment the HLA class I peptides consist of or comprise SEQ ID NO:6,7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLAclass II peptide consists of or comprises SEQ ID NO:8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and55, and bind to the corresponding HLA molecule or molecules indicatedfor each peptide in the tables below. In one embodiment, certainpeptides can bind to more than one HLA allele.

In another embodiment, a modification of a peptide of the invention isprovided. In one embodiment the modification comprises at least oneheteroclitic amino acid change, also referred to as a mutation ormutated, or an anchor residue mutation (see below). An HLA class Imolecule binding motif of a modified peptide of the present inventionexhibits an increased affinity for the HLA class I molecule, relative tothe unmutated counterpart of the peptide. In another embodiment, thepoint mutation increases the affinity of the isolated, mutatedWT1-derived peptide for the HLA class I molecule. In another embodiment,the increase in affinity is relative to the affinity (for the same HLAclass I molecule) of the isolated, unmutated WT1-derived peptidewherefrom the isolated, mutated WT1-derived peptide was derived. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a WT1 peptide of methods and compositions of thepresent invention is so designed as to exhibit affinity for an HLAmolecule. In another embodiment, the affinity is a high affinity, asdescribed herein.

HLA molecules, known in another embodiment as major histocompatibilitycomplex (MHC) molecules, bind peptides and present them to immune cells.Thus, in another embodiment, the immunogenicity of a peptide ispartially determined by its affinity for HLA molecules. HLA class Imolecules interact with CD8 molecules, which are generally present oncytotoxic T lymphocytes (CTL). HLA class II molecules interact with CD4molecules, which are generally present on helper T lymphocytes.

In another embodiment, a peptide of the present invention isimmunogenic. In another embodiment, “immunogenic” refers to an abilityto stimulate, elicit or participate in an immune response. In anotherembodiment, the immune response elicited is a cell-mediated immuneresponse. In another embodiment, the immune response is a combination ofcell-mediated and humoral responses.

In another embodiment, T cells that bind to the MHC molecule-peptidecomplex become activated and induced to proliferate and lyse cellsexpressing a protein comprising the peptide. T cells are typicallyinitially activated by “professional” antigen presenting cells (“APC”;e.g. dendritic cells, monocytes, and macrophages), which presentcostimulatory molecules that encourage T cell activation as opposed toanergy or apoptosis. In another embodiment, the response isheteroclitic, as described herein, such that the CTL lyses a neoplasticcell expressing a protein which has an AA sequence homologous to apeptide of this invention, or a different peptide than that used tofirst stimulate the T cell.

In another embodiment, an encounter of a T cell with a peptide of thisinvention induces its differentiation into an effector and/or memory Tcell. Subsequent encounters between the effector or memory T cell andthe same peptide, or, in another embodiment, with a related peptide ofthis invention, leads to a faster and more intense immune response. Suchresponses are gauged, in another embodiment, by measuring the degree ofproliferation of the T cell population exposed to the peptide. Inanother embodiment, such responses are gauged by any of the methodsenumerated hereinbelow.

In another embodiment, the peptides of methods and compositions of thepresent invention bind an HLA class II molecule with high affinity. Inother embodiments, the HLA class II molecule is any HLA class IImolecule enumerated herein. Each possibility represents a separateembodiment of the present invention.

In another embodiment, derivatives of peptides of methods andcompositions of the present invention bind an HLA class I molecule withhigh affinity. In other embodiments, the MHC class I molecule is any MHCclass I molecule enumerated herein. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a peptide of methods and compositions of thepresent invention binds an HLA class II molecule with significantaffinity, while a peptide derived from the original peptide binds an HLAclass I molecule with significant affinity.

In another embodiment, “affinity” refers to the concentration of peptidenecessary for inhibiting binding of a standard peptide to the indicatedMHC molecule by 50%. In another embodiment, “high affinity” refers to anaffinity is such that a concentration of about 500 nanomolar (nM) orless of the peptide is required for 50% inhibition of binding of astandard peptide. In another embodiment, a concentration of about 400 nMor less of the peptide is required. In another embodiment, the bindingaffinity is 300 nM. In another embodiment, the binding affinity is 200nM. In another embodiment, the binding affinity is 150 nM. In anotherembodiment, the binding affinity is 100 nM. In another embodiment, thebinding affinity is 80 nM. In another embodiment, the binding affinityis 60 nM. In another embodiment, the binding affinity is 40 nM. Inanother embodiment, the binding affinity is 30 nM. In anotherembodiment, the binding affinity is 20 nM. In another embodiment, thebinding affinity is 15 nM. In another embodiment, the binding affinityis 10 nM. In another embodiment, the binding affinity is 8 nM. Inanother embodiment, the binding affinity is 6 nM. In another embodiment,the binding affinity is 4 nM. In another embodiment, the bindingaffinity is 3 nM. In another embodiment, the binding affinity is 2 nM.In another embodiment, the binding affinity is 1.5 nM. In anotherembodiment, the binding affinity is 1 nM. In another embodiment, thebinding affinity is 0.8 nM. In another embodiment, the binding affinityis 0.6 nM. In another embodiment, the binding affinity is 0.5 nM. Inanother embodiment, the binding affinity is 0.4 nM. In anotherembodiment, the binding affinity is 0.3 nM. In another embodiment, thebinding affinity is less than 0.3 nM.

In another embodiment, “affinity” refers to a measure of bindingstrength to the MHC molecule. In another embodiment, affinity ismeasured using a method known in the art to measure competitive bindingaffinities. In another embodiment, affinity is measured using a methodknown in the art to measure relative binding affinities. In anotherembodiment, the method is a competitive binding assay. In anotherembodiment, the method is radioimmunoassay or RIA. In anotherembodiment, the method is BiaCore analyses. In another embodiment, themethod is any other method known in the art. In another embodiment, themethod yields an IC50 in relation to an IC50 of a reference peptide ofknown affinity.

Each type of affinity and method of measuring affinity represents aseparate embodiment of the present invention.

In another embodiment, “high affinity” refers to an IC50 of 0.5-500 nM.In another embodiment, the IC50 is 1-300 nM. In another embodiment, theIC50 is 1.5-200 nM. In another embodiment, the IC50 is 2-100 nM. Inanother embodiment, the IC50 is 3-100 nM. In another embodiment, theIC50 is 4-100 nM. In another embodiment, the IC50 is 6-100 nM. Inanother embodiment, the IC50 is 10-100 nM. In another embodiment, theIC50 is 30-100 nM. In another embodiment, the IC50 is 3-80 nM. Inanother embodiment, the IC50 is 4-60 nM. In another embodiment, the IC50is 5-50 nM. In another embodiment, the IC50 is 6-50 nM. In anotherembodiment, the IC50 is 8-50 nM. In another embodiment, the IC50 is10-50 nM. In another embodiment, the IC50 is 20-50 nM. In anotherembodiment, the IC50 is 6-40 nM. In another embodiment, the IC50 is 8-30nM. In another embodiment, the IC50 is 10-25 nM. In another embodiment,the IC50 is 15-25 nM. Each affinity and range of affinities represents aseparate embodiment of the present invention.

In another embodiment, a peptide of methods and compositions of thepresent invention binds to a superfamily of HLA molecules. Superfamiliesof HLA molecules share very similar or identical binding motifs. Inanother embodiment, the superfamily is a HLA class I superfamily. Inanother embodiment, the superfamily is a HLA class II superfamily. Eachpossibility represents a separate embodiment of the present invention.

The terms “HLA-binding peptide,” “HLA class I molecule-binding peptide,”and “HLA class II molecule-binding peptide” refer, in anotherembodiment, to a peptide that binds an HLA molecule with measurableaffinity. In another embodiment, the terms refer to a peptide that bindsan HLA molecule with high affinity. In another embodiment, the termsrefer to a peptide that binds an HLA molecule with sufficient affinityto activate a T cell precursor. In another embodiment, the terms referto a peptide that binds an HLA molecule with sufficient affinity tomediate recognition by a T cell. The HLA molecule is, in otherembodiments, any of the HLA molecules enumerated herein. Eachpossibility represents a separate embodiment of the present invention.

“Heteroclitic” refers, in another embodiment, to a peptide thatgenerates an immune response that recognizes the original peptide fromwhich the heteroclitic peptide was derived (e.g. the peptide notcontaining the anchor residue or other residue mutations). In anotherembodiment, “original peptide” refers to a peptide of the presentinvention. In another embodiment, “heteroclitic” refers to a peptidethat generates an immune response that recognizes the original peptidefrom which the heteroclitic peptide was derived, wherein the immuneresponse generated by vaccination with the heteroclitic peptide isgreater than the immune response generated by vaccination with theoriginal peptide. In another embodiment, a “heteroclitic” immuneresponse refers to an immune response that recognizes the originalpeptide from which the improved peptide was derived (e.g. the peptidenot containing the anchor residue mutations). In another embodiment, a“heteroclitic” immune response refers to an immune response thatrecognizes the original peptide from which the heteroclitic peptide wasderived, wherein the magnitude of the immune response generated byvaccination with the heteroclitic peptide is greater than the immuneresponse generated by vaccination with the original peptide. In anotherembodiment, the magnitude of the immune response generated byvaccination with the heteroclitic peptide is greater than the immuneresponse substantially equal to the response to vaccination with theoriginal peptide. In another embodiment, the magnitude of the immuneresponse generated by vaccination with the heteroclitic peptide isgreater than the immune response less than the response to vaccinationwith the original peptide. In another embodiment, a heteroclitic peptideof the present invention is an HLA class I heteroclitic peptide. Methodsfor identifying HLA class I and class II residues, and for improving HLAbinding by mutating the residues, are well known in the art, asdescribed below. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, a heteroclitic peptide of the present inventioninduces an immune response that is increased at least 2-fold relative tothe WT1 peptide from which the heteroclitic peptide was derived (“nativepeptide”). In another embodiment, the increase is 3-fold relative to thenative peptide. In another embodiment, the increase is 5-fold relativeto the native peptide. In another embodiment, the increase is 7-foldrelative to the native peptide. In another embodiment, the increase is10-fold relative to the native peptide. In another embodiment, theincrease is 15-fold relative to the native peptide. In anotherembodiment, the increase is 20-fold relative to the native peptide. Inanother embodiment, the increase is 30-fold relative to the nativepeptide. In another embodiment, the increase is 50-fold relative to thenative peptide. In another embodiment, the increase is 100-fold relativeto the native peptide. In another embodiment, the increase is 150-foldrelative to the native peptide. In another embodiment, the increase is200-fold relative to the native peptide. In another embodiment, theincrease is 300-fold relative to the native peptide. In anotherembodiment, the increase is 500-fold relative to the native peptide. Inanother embodiment, the increase is 1000-fold relative to the nativepeptide. In another embodiment, the increase is more than 1000-foldrelative to the native peptide. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a HLA class IIheteroclitic peptide derived from an isolated WT1 peptide of the presentinvention. In another embodiment, the process of deriving comprisesintroducing a mutation that enhances a binding of the peptide to an HLAclass II molecule. In another embodiment, the process of derivingconsists of introducing a mutation that enhances a binding of thepeptide to an HLA class I molecule. In another embodiment, the mutationis in an HLA class II anchor residue. In another embodiment, aheteroclitic class II peptide of the present invention is identified andtested in a manner analogous to identification and testing of HLA classI heteroclitic peptides, as exemplified herein. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the HLA class II binding site in a peptide of thepresent invention is created or improved by mutation of an HLA class IImotif anchor residue. In another embodiment, the anchor residue that ismodified is in the P1 position. In another embodiment, the anchorresidue is at the P2 position. In another embodiment, the anchor residueis at the P6 position. In another embodiment, the anchor residue is atthe P9 position. In another embodiment, the anchor residue is selectedfrom the P1, P2, P6, and P9 positions. In another embodiment, the anchorresidue is at the P3 position. In another embodiment, the anchor residueis at the P4 position. In another embodiment, the anchor residue is atthe P5 position. In another embodiment, the anchor residue is at the P6position. In another embodiment, the anchor residue is at the P8position. In another embodiment, the anchor residue is at the P10position. In another embodiment, the anchor residue is at the P1 1position. In another embodiment, the anchor residue is at the P12position. In another embodiment, the anchor residue is at the P13position. In another embodiment, the anchor residue is at any otheranchor residue of an HLA class II molecule that is known in the art. Inanother embodiment, residues other than P1, P2, P6, and P9 serve assecondary anchor residues; therefore, mutating them can improve HLAclass II binding. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, a heteroclitic peptide is generated byintroduction of a mutation that creates an anchor motif. “Anchor motifs”or “anchor residues” refers, in another embodiment, to 1 or a set ofpreferred residues at particular positions in an HLA-binding sequence.In another embodiment, the

HLA-binding sequence is an HLA class II-binding sequence. In anotherembodiment, the HLA-binding sequence is an HLA class I-binding sequence.In another embodiment, the positions corresponding to the anchor motifsare those that play a significant role in binding the HLA molecule. Inanother embodiment, the anchor residue is a primary anchor motif. Inanother embodiment, the anchor residue is a secondary anchor motif. Eachpossibility represents a separate embodiment of the present invention.

Methods for predicting MHC class I and II epitopes are well known in theart. In one embodiment, the software of the Bioinformatics & MolecularAnalysis Section (National Institutes of Health, Washington, D.C.)available at http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken parkercomboform is useful. This software ranks 9-mer or 10-mer peptides on apredicted half-time dissociation coefficient from HLA class I molecules(Pinilla, et al. Curr Opin Immunol, 11 (2): p. 193-202 (1999)). Inanother embodiment, MHC class II epitope is predicted using TEPITOPE(Meister G E, Roberts C G et al, Vaccine 1995 13: 581-91). In anotherembodiment, the MHC class II epitope is predicted using EpiMatrix (DeGroot A S, Jesdale B M et al, AIDS Res Hum Retroviruses 1997 13:529-31). In another embodiment, the MHC class II epitope is predictedusing the Predict Method (Yu K, Petrovsky N et al, Mol Med. 2002 8:137-48). In another embodiment, the MHC class II epitope is predictedusing the SYFPEITHI epitope prediction algorithm (Examples). In anotherembodiment, the MHC class II epitope is predicted using Rankpep. Inanother embodiment, the MHC class II epitope is predicted using anyother method known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, in the case of HLA class II-binding peptides(e.g. HLA-DR-binding peptides), the anchor residue that is modified isin the P1 position. In another embodiment, the anchor residue is in theP2 position. In another embodiment, the anchor residue is in the P6position. In another embodiment, the anchor residue is in the P9position. In other embodiments, the anchor residue is the P3, P4, P5,P6, P8, P10, P11, P12, or P13 position. In another embodiment, theanchor residue is any other anchor residue of an HLA class II moleculethat is known in the art. In another embodiment, residues other than P1,P2, P6, and P9 serve as secondary anchor residues; therefore, mutatingthem can improve HLA class II binding. In another embodiment, anycombination of the above residues is mutated. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, a WT1 peptide of the present invention binds to 2distinct HLA class II molecules. In another embodiment, the peptidebinds to three distinct HLA class II molecules. In another embodiment,the peptide binds to four distinct HLA class II molecules. In anotherembodiment, the peptide binds to five distinct HLA class II molecules.In another embodiment, the peptide binds to six distinct HLA class IImolecules. In another embodiment, the peptide binds to more than sixdistinct HLA class II molecules.

In another embodiment, the HLA class II molecules that are bound by aWT1 peptide of the present invention are encoded by two or more distinctalleles at a given HLA class II locus. In another embodiment, the HLAclass II molecules are encoded by 3 distinct alleles at a locus. Inanother embodiment, the HLA class II molecules are encoded by 4 distinctalleles at a locus. In another embodiment, the HLA class II moleculesare encoded by 5 distinct alleles at a locus. In another embodiment, theHLA class II molecules are encoded by 6 distinct alleles at a locus. Inanother embodiment, the HLA class II molecules are encoded by more thansix distinct alleles at a locus.

In another embodiment, the HLA class II molecules bound by the WT1peptide are encoded by HLA class II genes at 2 distinct loci. In anotherembodiment, the HLA molecules bound are encoded by HLA class II genes at2 or more distinct loci. In another embodiment, the HLA molecules boundare encoded by HLA class II genes at 3 distinct loci. In anotherembodiment, the HLA molecules bound are encoded by HLA class II genes at3 or more distinct loci. In another embodiment, the HLA molecules boundare encoded by HLA class II genes at 4 distinct loci. In anotherembodiment, the HLA molecules bound are encoded by HLA class II genes at4 or more distinct loci. In another embodiment, the HLA molecules boundare encoded by HLA class II genes at more than 4 distinct loci. In otherembodiments, the loci are selected from HLA-DRB loci. In anotherembodiment, the HLA class II-binding peptide is an HLA-DRA bindingpeptide. In another embodiment, the peptide is an HLA-DQA1 bindingpeptide. In another embodiment, the peptide is an HLA-DQB 1 bindingpeptide. In another embodiment, the peptide is an HLA-DPA1 bindingpeptide. In another embodiment, the peptide is an HLA-DPB 1 bindingpeptide. In another embodiment, the peptide is an HLA-DMA bindingpeptide. In another embodiment, the peptide is an HLA-DMB bindingpeptide. In another embodiment, the peptide is an HLA-DOA bindingpeptide. In another embodiment, the peptide is an HLA-DOB bindingpeptide. In another embodiment, the peptide binds to any other HLA classII molecule known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a WT1 peptide of the present invention binds to 2distinct HLA-DRB molecules. In another embodiment, the peptide binds to3 distinct HLA-DRB molecules. In another embodiment, the peptide bindsto 4 distinct HLA-DRB molecules. In another embodiment, the peptidebinds to 5 distinct HLA-DRB molecules. In another embodiment, thepeptide binds to 6 distinct HLA-DRB molecules. In another embodiment,the peptide binds to more than 6 distinct HLA-DRB molecules.

In another embodiment, a WT1 peptide of the present invention binds toHLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles. Inanother embodiment, the HLA-DRB molecules are encoded by 3 distinctHLA-DRB alleles. In another embodiment, the HLA-DRB molecules areencoded by 4 distinct HLA-DRB alleles. In another embodiment, theHLA-DRB molecules are encoded by 5 distinct HLA-DRB alleles. In anotherembodiment, the HLA-DRB molecules are encoded by 6 distinct HLA-DRBalleles. In another embodiment, the HLA-DRB molecules are encoded bymore than 6 distinct HLA-DRB alleles. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a WT1 peptide of the present invention binds toHLA-DRB molecules that are encoded by 2 distinct HLA-DRB allelesselected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB1501. In another embodiment, the WT1 peptide binds to HLA-DRB moleculesencoded by 3 distinct HLA-DRB alleles selected from DRB 101, DRB 301,DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1peptide binds to HLA-DRB molecules encoded by 4 distinct HLA-DRB allelesselected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB1501. In another embodiment, the WT1 peptide binds to HLA-DRB moleculesencoded by 5 distinct HLA-DRB alleles selected from DRB 101, DRB 301,DRB 401, DRB 701, DRB 1101, DRB 1104 and DRB 1501. In anotherembodiment, the WT1 peptide binds to HLA-DRB molecules encoded by eachof the following HLA-DRB alleles: DRB 101, DRB 301, DRB 401, DRB 701,DRB 1101, and DRB 1501. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a compositioncomprising 2 distinct WT1 peptides of the present invention. In anotherembodiment, the 2 distinct WT1 peptides are both unaltered. In anotherembodiment, 1 of the WT1 peptides is unaltered, while the other isheteroclitic. In another embodiment, both of the WT1 peptides areheteroclitic.

In another embodiment, the composition comprises 3 distinct WT1 peptidesof the present invention. In another embodiment, the compositioncomprises 4 distinct WT1 peptides of the present invention. In anotherembodiment, the composition comprises 5 distinct WT1 peptides of thepresent invention. In another embodiment, the composition comprises morethan 5 distinct isolated WT1 peptides of the present invention.

In another embodiment, 2 of the WT1 peptides in the composition areunaltered. In another embodiment, 2 of the WT1 peptides in thecomposition are heteroclitic. In another embodiment, 2 of the WT1peptides in the composition are unaltered, and 2 are heteroclitic. Inanother embodiment, more than 2 of the WT1 peptides in the compositionare unaltered. In another embodiment, more than 2 of the WT1 peptides inthe composition are heteroclitic. In another embodiment, more than 2 ofthe WT1 peptides in the composition are unaltered, and more than 2 areheteroclitic. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, 1 of the additional WT1 peptides in a compositionof the present invention has a sequence selected from the sequences setforth in SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46,47, 48, 49, 50 and 55. In another embodiment, 2 of the additional WT1peptides have a sequence selected from the sequences set forth in SEQ IDNo: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50and 55. In another embodiment, 3 of the additional WT1 peptides have asequence selected from the sequences set forth in SEQ ID No: 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33,35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55.

In another embodiment, any other immunogenic WT1 peptide known in theart is utilized as an additional WT1 peptide. In another embodiment, anycombination of immunogenic WT1 peptides known in the art is utilized.Non-limiting sources of other WT1 peptides include WO2005053618,WO2007047764 and WO2007120673.

Each additional WT1 peptide, and each combination thereof, represents aseparate embodiment of the present invention.

In another embodiment, a composition of the present invention contains 2HLA class II heteroclitic peptides that are derived from the sameisolated WT1 peptide of the present invention. In another embodiment,the 2 HLA class II heteroclitic peptides contain mutations in differentHLA class II molecule anchor residues. In another embodiment, the 2 HLAclass II heteroclitic peptides contain different mutations in the sameanchor residues. In another embodiment, 2 of the HLA class IIheteroclitic peptides are derived from different isolated WT1 peptidesof the present invention. Each possibility represents a separateembodiment of the present invention.

In another embodiment, 2 WT1 peptides of the present invention, or theWT1 peptides that correspond to two HLA class II heteroclitic peptidesof the present invention, overlap with one another. In anotherembodiment, the overlap between the peptides is at least 7 amino acids(AA). In another embodiment, the overlap is at least 8 AA. In anotherembodiment, the overlap is at least 9 AA. In another embodiment, theoverlap is 7 AA. In another embodiment, the overlap is 8 AA. In anotherembodiment, the overlap is 9 AA. In another embodiment, the overlap is10 AA. In another embodiment, the overlap is 11 AA. In anotherembodiment, the overlap is 12 AA. In another embodiment, the overlap is13 AA. In another embodiment, the overlap is 14 AA. In anotherembodiment, the overlap is 15 AA. In another embodiment, the overlap is16 AA. In another embodiment, the overlap is more than 16 AA. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the peptides in a composition of the presentinvention bind to 2 distinct HLA class II molecules. In anotherembodiment, the peptides bind to 3 distinct HLA class II molecules. Inanother embodiment, the peptides bind to 4 distinct HLA class IImolecules. In another embodiment, the peptides bind to 5 distinct HLAclass II molecules. In another embodiment, the peptides bind to morethan 5 distinct HLA class II molecules. In another embodiment, thepeptides in the composition bind to the same HLA class II molecules.

In another embodiment, each of the WT 1 peptides in a composition of thepresent invention binds to a set of HLA class II molecules. In anotherembodiment, each of the WT1 peptides binds to a distinct set of HLAclass II molecules. In another embodiment, the WT1 peptides in thecomposition bind to the same set of HLA class II molecules. In anotherembodiment, 2 of the WT1 peptides bind to a distinct but overlapping setof HLA class II molecules. In another embodiment, 2 or more of the WT1peptides bind to the same set of HLA class II molecules, while anotherof the WT1 peptides binds to a distinct set. In another embodiment, 2 ormore of the WT1 peptides bind to an overlapping set of HLA class IImolecules, while another of the WT1 peptides binds to a distinct set.

In another embodiment, 2 or more of the WT1 peptides in a composition ofthe present invention each binds to more than 1 HLA-DRB molecule. Inanother embodiment, the 4 or more HLA-DRB molecules bound by thepeptides in the composition are distinct from one another. In anotherembodiment, the HLA-DRB molecules are encoded by different HLA-DRBalleles. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, 2 or more of the HLA class II molecules bound byWT1 peptides in a composition of the present invention are HLA-DRBmolecules. In another embodiment, 3 or more of the HLA class IImolecules that are bound are HLA-DRB molecules. In other embodiments,the HLA class II molecules that are bound can be any of the HLA class IImolecules enumerated herein. In another embodiment, the HLA class IImolecules that are bound are encoded by 2 or more distinct HLA class IIalleles at a given locus. In another embodiment, the HLA class IImolecules that are bound are encoded by HLA class II genes at 2 or moredistinct loci.

Each of the above compositions represents a separate embodiment of thepresent invention.

In another embodiment, a “set of HLA class II molecules” refers to theHLA class II molecules encoded by different alleles at a particularlocus. In another embodiment, the term refers to HLA class II moleculeswith a particular binding specificity. In another embodiment, the termrefers to HLA class II molecules with a particular peptide consensussequence. In another embodiment, the term refers to a superfamily of HLAclass II molecules. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a compositioncomprising an unaltered HLA class II molecule-binding WT1 peptide of thepresent invention and a second, HLA class I molecule-binding WT1peptide. In another embodiment, the composition comprises more than 1HLA class II molecule-binding WT1 peptide of the present invention, inaddition to the HLA class I molecule-binding WT1 peptide. In anotherembodiment, the composition comprises more than 1 HLA class Imolecule-binding WT1 peptide, in addition to the HLA class IImolecule-binding WT1 peptide. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the AA sequence of the HLA class Imolecule-binding WT1 peptide comprises a sequence selected from SEQ IDNo: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50and 55. In another embodiment, the AA sequence of the HLA class Imolecule-binding WT1 peptide is selected from the sequences set forth inSEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48,49, 50 and 55. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the HLA class I molecule-binding WT1 peptide isan HLA class I heteroclitic peptide. In another embodiment, the HLAclass I molecule-binding WT1 peptide contains a mutation in an HLA classI molecule anchor residue thereof, as described further herein. Asprovided herein, WT1-derived peptides were modified in HLA anchorresidues to generate heteroclitic peptides with increased predictedbinding to HLA-A0201 and HLA-A0301. Peptides with increased predictedbinding also exhibited enhanced ability to bind HLA class I moleculesand increased immunogenicity.

In another embodiment, the mutation that enhances MHC binding is in theresidue at position 1 of the HLA class I heteroclitic peptide. Inanother embodiment, the residue is changed to tyrosine. In anotherembodiment, the residue is changed to glycine. In another embodiment,the residue is changed to threonine. In another embodiment, the residueis changed to phenylalanine. In another embodiment, the residue ischanged to any other residue known in the art. In another embodiment, asubstitution in position 1 (e.g. to tyrosine) stabilizes the binding ofthe position 2 anchor residue.

In another embodiment, the mutation is in position 2 of the HLA class Iheteroclitic peptide. In another embodiment, the residue is changed toleucine. In another embodiment, the residue is changed to valine. Inanother embodiment, the residue is changed to isoleucine. In anotherembodiment, the residue is changed to methionine. In another embodiment,the residue is changed to any other residue known in the art.

In another embodiment, the mutation is in position 6 of the HLA class Iheteroclitic peptide. In another embodiment, the residue is changed tovaline. In another embodiment, the residue is changed to cysteine. Inanother embodiment, the residue is changed to glutamine. In anotherembodiment, the residue is changed to histidine. In another embodiment,the residue is changed to any other residue known in the art.

In another embodiment, the mutation is in position 9 of the HLA class Iheteroclitic peptide. In another embodiment, the mutation changes theresidue at the C-terminal position thereof. In another embodiment, theresidue is changed to valine. In another embodiment, the residue ischanged to threonine. In another embodiment, the residue is changed toisoleucine. In another embodiment, the residue is changed to leucine. Inanother embodiment, the residue is changed to alanine. In anotherembodiment, the residue is changed to cysteine. In another embodiment,the residue is changed to any other residue known in the art.

In another embodiment, the point mutation is in a primary anchorresidue. In another embodiment, the HLA class I primary anchor residuesare positions 2 and 9. In another embodiment, the point mutation is in asecondary anchor residue. In another embodiment, the HLA class Isecondary anchor residues are positions 1 and 8. In another embodiment,the HLA class I secondary anchor residues are positions 1, 3, 6, 7, and8. In another embodiment, the point mutation is in a position selectedfrom positions 4, 5, and 8. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the point mutation is in 1 or more residues inpositions selected from positions 1, 2, 8, and 9 of the HLA class Ibinding motif. In another embodiment, the point mutation is in 1 or moreresidues in positions selected from positions 1, 3, 6, and 9. In anotherembodiment, the point mutation is in 1 or more residues in positionsselected from positions 1, 2, 6, and 9. In another embodiment, the pointmutation is in 1 or more residues in positions selected from positions1, 6, and 9. In another embodiment, the point mutation is in 1 or moreresidues in positions selected from positions 1, 2, and 9. In anotherembodiment, the point mutation is in 1 or more residues in positionsselected from positions 1, 3, and 9. In another embodiment, the pointmutation is in 1 or more residues in positions selected from positions 2and 9. In another embodiment, the point mutation is in 1 or moreresidues in positions selected from positions 6 and 9. Each possibilityrepresents a separate embodiment of the present invention.

Each of the above anchor residues and substitutions represents aseparate embodiment of the present invention.

In another embodiment, the HLA class I molecule-binding WT peptide haslength of 9 AA. In another embodiment, the peptide has length of 10 AA.As provided herein, native and heteroclitic peptides of 9-10 AAexhibited substantial binding to HLA class I molecules and ability toelicit cytokine secretion and cytolysis by CTL.

In another embodiment, the HLA class I molecule that is bound by the HLAclass I molecule-binding WT1 peptide is an HLA-A molecule. In anotherembodiment, the HLA class I-molecule is an HLA-A2 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-A3 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-A1 1 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-B 8 molecule. In anotherembodiment, the HLA class I-molecule is an HLA-0201 molecule. In anotherembodiment, the HLA class I-molecule binds any other HLA class Imolecule known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a WT1 peptide of methods and compositions of thepresent invention has a length of 8-30 amino acids. In anotherembodiment, the peptide has a length of 9-11 AA. In another embodiment,the peptide ranges in size from 7-25 AA, or in another embodiment, 8-11,or in another embodiment, 8-15, or in another embodiment, 9-20, or inanother embodiment, 9-18, or in another embodiment, 9-15, or in anotherembodiment, 8-12, or in another embodiment, 9-11 AA in length. Inanother embodiment, the peptide is 8 AA in length, or in anotherembodiment, 9 AA or in another embodiment, 10 AA or in anotherembodiment, 12 AA or in another embodiment, 25 AA in length, or inanother embodiment, any length therebetween. In another embodiment, thepeptide is of greater length, for example 50, or 100, or more. In thisembodiment, the cell processes the peptide to a length of 7 and 25 AA inlength. In this embodiment, the cell processes the peptide to a lengthof 9-11 AA Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the peptide is 15-23 AA in length. In anotherembodiment, the length is 15-24 AA. In another embodiment, the length is15-25 AA. In another embodiment, the length is 15-26 AA. In anotherembodiment, the length is 15-27 AA. In another embodiment, the length is15-28 AA. In another embodiment, the length is 14-30 AA. In anotherembodiment, the length is 14-29 AA. In another embodiment, the length is14-28 AA. In another embodiment, the length is 14-26 AA. In anotherembodiment, the length is 14-24 AA. In another embodiment, the length is14-22 AA. In another embodiment, the length is 14-20 AA. In anotherembodiment, the length is 16-30 AA. In another embodiment, the length is16-28 AA. In another embodiment, the length is 16-26 AA. In anotherembodiment, the length is 16-24 AA. In another embodiment, the length is16-22 AA. In another embodiment, the length is 18-30 AA. In anotherembodiment, the length is 18-28 AA. In another embodiment, the length is18-26 AA. In another embodiment, the length is 18-24 AA. In anotherembodiment, the length is 18-22 AA. In another embodiment, the length is18-20 AA. In another embodiment, the length is 20-30 AA. In anotherembodiment, the length is 20-28 AA. In another embodiment, the length is20-26 AA. In another embodiment, the length is 20-24 AA. In anotherembodiment, the length is 22-30 AA. In another embodiment, the length is22-28 AA. In another embodiment, the length is 22-26 AA. In anotherembodiment, the length is 24-30 AA. In another embodiment, the length is24-28 AA. In another embodiment, the length is 24-26 AA.

Each of the above peptides, peptide lengths, and types of peptidesrepresents a separate embodiment of the present invention.

In another embodiment, minor modifications are made to peptides of thepresent invention without decreasing their affinity for HLA molecules orchanging their TCR specificity, utilizing principles well known in theart. In the case of HLA class I-binding peptides, “minor modifications”refers, in another embodiment, to e.g. insertion, deletion, orsubstitution of one AA, inclusive, or deletion or addition of 1-3 AAoutside of the residues between 2 and 9, inclusive. While the computeralgorithms described herein are useful for predicting the MHC classI-binding potential of peptides, they have 60-80% predictive accuracy;and thus, the peptides should be evaluated empirically before a finaldetermination of MHC class I-binding affinity is made. Thus, peptides ofthe present invention are not limited to peptides predicated by thealgorithms to exhibit strong MHC class I-binding affinity. The types aremodifications that can be made are listed below. Each modificationrepresents a separate embodiment of the present invention.

In another embodiment, a peptide enumerated in the Examples of thepresent invention is further modified by mutating an anchor residue toan MHC class I preferred anchor residue, which can be, in otherembodiments, any of the anchor residues enumerated herein. In anotherembodiment, a peptide of the present invention containing an MHC class Ipreferred anchor residue is further modified by mutating the anchorresidue to a different MHC class I preferred residue for that location.The different preferred residue can be, in other embodiments, any of thepreferred residues enumerated herein.

In another embodiment, the anchor residue that is further modified is inthe 1 position. In another embodiment, the anchor residue is in the 2position. In another embodiment, the anchor residue is in the 3position. In another embodiment, the anchor residue is in the 4position. In another embodiment, the anchor residue is in the 5position. In another embodiment, the anchor residue is in the 6position. In another embodiment, the anchor residue is in the 7position. In another embodiment, the anchor residue is in the 8position. In another embodiment, the anchor residue is in the 9position. In the case of HLA class I-binding peptides, residues otherthan 2 and 9 can serve as secondary anchor residues; therefore, mutatingthem can improve MHC class I binding. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, a peptide of methods and compositions of thepresent invention is a length variant of a peptide enumerated in theExamples. In another embodiment, the length variant is one amino acid(AA) shorter than the peptide from the Examples. In another embodiment,the length variant is two AA shorter than the peptide from the Examples.In another embodiment, the length variant is more than two AA shorterthan the peptide from the Examples. In another embodiment, the shorterpeptide is truncated on the N-terminal end. In another embodiment, theshorter peptide is truncated on the C-terminal end. In anotherembodiment, the truncated peptide is truncated on both the N-terminaland C-terminal ends. Peptides are, in another embodiment, amenable totruncation without changing affinity for HLA molecules, as is well knownin the art.

Each of the above truncated peptides represents a separate embodiment ofthe present invention.

In another embodiment, the length variant is longer than a peptideenumerated in the Examples of the present invention. In anotherembodiment, the longer peptide is extended on the N-terminal end inaccordance with the surrounding WT1 sequence. Peptides are, in anotherembodiment, amenable to extension on the N-terminal end without changingaffinity for HLA molecules, as is well known in the art. Such peptidesare thus equivalents of the peptides enumerated in the Examples. Inanother embodiment, the N-terminal extended peptide is extended by oneresidue. In another embodiment, the N-terminal extended peptide isextended by two residues. In another embodiment, the N-terminal extendedpeptide is extended by three residues. In another embodiment, theN-terminal extended peptide is extended by more than three residues.

In another embodiment, the longer peptide is extended on the C terminalend in accordance with the surrounding WT1 sequence. Peptides are, inanother embodiment, amenable to extension on the C-terminal end withoutchanging affinity for HLA molecules, as is well known in the art. Suchpeptides are thus equivalents of the peptides enumerated in the Examplesof the present invention. In another embodiment, the C-terminal extendedpeptide is extended by one residue. In another embodiment, theC-terminal extended peptide is extended by two residues. In anotherembodiment, the C-terminal extended peptide is extended by threeresidues. In another embodiment, the C-terminal extended peptide isextended by more than three residues.

In another embodiment, the extended peptide is extended on both theN-terminal and C-terminal ends in accordance with the surrounding WT1sequence.

Each of the above extended peptides represents a separate embodiment ofthe present invention.

In another embodiment, a truncated peptide of the present inventionretains the HLA anchor residues (e.g. the HLA class I anchor residues)on the second residue and the C-terminal residue, with a smaller numberof intervening residues (e.g., 5) than a peptide enumerated in theExamples of the present invention. Peptides are, in another embodiment,amenable to such mutation without changing affinity for

HLA molecules. In another embodiment, such a truncated peptide isdesigned by removing one of the intervening residues of one of the abovesequences. In another embodiment, the HLA anchor residues are retainedon the second and eighth residues. In another embodiment, the HLA anchorresidues are retained on the first and eighth residues. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, an extended peptide of the present inventionretains the HLA anchor residues (e.g. the HLA class I anchor residues)on the second residue and the C-terminal residue, with a larger numberof intervening residues (e.g. 7 or 8) than a peptide enumerated in theExamples of the present invention. In another embodiment, such anextended peptide is designed by adding one or more residues between twoof the intervening residues of one of the above sequences. It is wellknown in the art that residues can be removed from or added between theintervening sequences of HLA-binding peptides without changing affinityfor HLA. Such peptides are thus equivalents of the peptides enumeratedin the Examples of the present invention. In another embodiment, the HLAanchor residues are retained on the second and ninth residues. Inanother embodiment, the HLA anchor residues are retained on the firstand eighth residues. In another embodiment, the HLA anchor residues areretained on the two residues separated by six intervening residues. Eachpossibility represents a separate embodiment of the present invention.

“Fragment,” in another embodiment, refers to a peptide of 11 or more AAin length. In another embodiment, a peptide fragment of the presentinvention is 16 or more AA long. In another embodiment, the fragment is12 or more AA long. In another embodiment, the fragment is 13 or moreAA. In another embodiment, the fragment is 14 or more AA. In anotherembodiment, the fragment is 15 or more AA. In another embodiment, thefragment is 17 or more AA. In another embodiment, the fragment is 18 ormore AA. In another embodiment, the fragment is 19 or more AA. Inanother embodiment, the fragment is 22 or more AA. In anotherembodiment, the fragment is 8-12 AA. In another embodiment, the fragmentis about 8-12 AA. In another embodiment, the fragment is 16-19 AA. Inanother embodiment, the fragment is about 16-19 AA. In anotherembodiment, the fragment 10-25 AA. In another embodiment, the fragmentis about 10-25 AA. In another embodiment, the fragment has any otherlength. Each possibility represents a separate embodiment of the presentinvention.

“Fragment of a WT1 protein,” in another embodiment, refers to any of thedefinitions of “fragment” found herein. Each definition represents aseparate embodiment of the present invention.

In another embodiment, a peptide of the present invention is homologousto a peptide enumerated in the Examples. The terms “homology,”“homologous,” etc., when in reference to any protein or peptide, refer,in another embodiment, to a percentage of amino acid residues in thecandidate sequence that are identical with the residues of acorresponding native polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Methods and computer programs for the alignment arewell known in the art.

In another embodiment, the term “homology,” when in reference to anynucleic acid sequence similarly indicates a percentage of nucleotides ina candidate sequence that are identical with the nucleotides of acorresponding native nucleic acid sequence.

Homology is, in another embodiment, determined by computer algorithm forsequence alignment, by methods well described in the art. In otherembodiments, computer algorithm analysis of nucleic acid sequencehomology includes the utilization of any number of software packagesavailable, such as, for example, the BLAST, DOMAIN, BEAUTY (BLASTEnhanced Alignment Utility), GENPEPT and TREMBL packages.

In another embodiment, “homology” refers to identity to a sequenceselected from SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44,46, 47, 48, 49, 50 and 55 of greater than 70%. In another embodiment,“homology” refers to identity to a sequence selected from SEQ ID No: 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31,32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 ofgreater than 72%. In another embodiment, “homology” refers to identityto one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46,47, 48, 49, 50 and 55 of greater than 75%. In another embodiment,“homology” refers to identity to a sequence selected from SEQ ID No: 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31,32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 ofgreater than 78%. In another embodiment, “homology” refers to identityto one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46,47, 48, 49, 50 and 55 of greater than 80%. In another embodiment,“homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36,37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than82%. In another embodiment, “homology” refers to identity to a sequenceselected from SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44,46, 47, 48, 49, 50 and 55 of greater than 83%. In another embodiment,“homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36,37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than85%. In another embodiment, “homology” refers to identity to one of SEQID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49,50 and 55 of greater than 87%. In another embodiment, “homology” refersto identity to a sequence selected from SEQ ID No: 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36,37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than[0128] 88%. In another embodiment, “homology” refers to identity to oneof SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47,48, 49, 50 and 55 of greater than 90%. In another embodiment, “homology”refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39,41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 92%. Inanother embodiment, “homology” refers to identity to a sequence selectedfrom SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47,48, 49, 50 and 55 of greater than 93%. In another embodiment, “homology”refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39,41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 95%. Inanother embodiment, “homology” refers to identity to a sequence selectedfrom SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47,48, 49, 50 and 55 of greater than 96%. In another embodiment, “homology”refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39,41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 97%. Inanother embodiment, “homology” refers to identity to one of SEQ ID No:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30,31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and55 of greater than 98%. In another embodiment, “homology” refers toidentity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42,43, 44, 46, 47, 48, 49, 50 and 55 of greater than 99%. In anotherembodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32,33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of100%. Each possibility represents a separate embodiment of the presentinvention. [00114] In another embodiment, homology is determined viadetermination of candidate sequence hybridization, methods of which arewell described in the art (See, for example, “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook etal., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, N. Y.; and Ausubel et al., 1989, Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N. Y). Inanother embodiments, methods of hybridization are carried out undermoderate to stringent conditions, to the complement of a DNA encoding anative caspase peptide. Hybridization conditions being, for example,overnight incubation at 42 <0>C in a solution comprising: 10-20%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20[mu]g/ml denatured, sheared salmon sperm DNA.

Each of the above homologues and variants of peptides enumerated in theExamples represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a compositioncomprising a peptide of this invention. In another embodiment, thecomposition further comprises a pharmaceutically acceptable carrier. Inanother embodiment, the composition further comprises an adjuvant. Inanother embodiment, the composition comprises 2 or more peptides of thepresent invention. In another embodiment, the composition furthercomprises any of the additives, compounds, or excipients set forthhereinbelow. In another embodiment, the adjuvant is KLH, QS21, Freund'scomplete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide,BCG or alum. In other embodiments, the carrier is any carrier enumeratedherein. In other embodiments, the adjuvant is any adjuvant enumeratedherein. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, this invention provides a vaccine comprising apeptide of this invention. In another embodiment, this inventionprovides a vaccine comprising an antigen-presenting cell (APC) and apeptide of this invention. In another embodiment, the vaccine furthercomprises a carrier. In another embodiment, the vaccine furthercomprises an adjuvant. In another embodiment, the vaccine furthercomprises an APC. In another embodiment, the vaccine further comprises acombination of more than 1 of an antigen, carrier, and/or APC. Inanother embodiment, the vaccine is a cell-based composition. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the term “vaccine” refers to a material orcomposition that, when introduced into a subject, provides aprophylactic or therapeutic response for a particular disease,condition, or symptom of same. In another embodiment, this inventioncomprises peptide-based vaccines, wherein the peptide comprises anyembodiment listed herein, including immunomodulating compounds such ascytokines, adjuvants, etc.

In another embodiment, a vaccine of methods and compositions of thepresent invention further comprises an adjuvant. In another embodiment,the adjuvant is Montanide ISA 51. Montanide ISA 51 contains a naturalmetabolizable oil and a refined emulsifier. In another embodiment, theadjuvant is GM-CSF. Recombinant GM-CSF is a human protein grown, inanother embodiment, in a yeast (S. cerevisiae) vector. GM-CSF promotesclonal expansion and differentiation of hematopoietic progenitor cells,APC, and dendritic cells and T cells.

In another embodiment, the adjuvant is a cytokine. In anotherembodiment, the adjuvant is a growth factor. In another embodiment, theadjuvant is a cell population. In another embodiment, the adjuvant isQS21. In another embodiment, the adjuvant is Freund's incompleteadjuvant. In another embodiment, the adjuvant is aluminum phosphate. Inanother embodiment, the adjuvant is aluminum hydroxide. In anotherembodiment, the adjuvant is BCG. In another embodiment, the adjuvant isalum.

In another embodiment, the adjuvant is an interleukin. In anotherembodiment, the adjuvant is a chemokine. In another embodiment, theadjuvant is any other type of adjuvant known in the art. In anotherembodiment, the WT1 vaccine comprises two the above adjuvants. Inanother embodiment, the WT1 vaccine comprises more than two the aboveadjuvants. Each possibility represents a separate embodiment of thepresent invention.

In other embodiments, a vaccine or composition of the present inventioncan comprise any of the embodiments of WT1 peptides of the presentinvention and combinations thereof. Each possibility represents aseparate embodiment of the present invention.

It is to be understood that any embodiments described herein, regardingpeptides, vaccines and compositions of this invention can be employed inany of the methods of this invention. Each combination of peptide,vaccine, or composition with a method represents an embodiment thereof.

In another embodiment, the present invention provides a method oftreating a subject with a WT1-expressing cancer, the method comprisingadministering to the subject a WT1 vaccine of the present invention,thereby treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a method oftreating a subject with an MDS, the method comprising administering tothe subject a WT1 vaccine of the present invention, thereby treating asubject with an MDS.

In another embodiment, the present invention provides a method ofsuppressing or halting the progression of a WT1-expressing cancer in asubject, the method comprising administering to the subject a WT1vaccine of the present invention, thereby suppressing or halting theprogression of a WT1-expressing cancer.

In another embodiment, the present invention provides a method ofreducing the incidence of a WT1-expressing cancer in a subject, themethod comprising administering to the subject a WT1 vaccine of thepresent invention, thereby reducing the incidence of a WT1-expressingcancer in a subject.

In another embodiment, the present invention provides a method ofreducing the incidence of an AML in a subject, the method comprisingadministering to the subject a WT1 vaccine of the present invention,thereby reducing the incidence of an AML.

In another embodiment, the present invention provides a method ofreducing the incidence of relapse of a WT1-expressing cancer in asubject, the method comprising administering to the subject a WT1vaccine of the present invention, thereby reducing the incidence ofrelapse of a WT1-expressing cancer in a subject.

In another embodiment, the present invention provides a method ofreducing the incidence of relapse of an AML in a subject, the methodcomprising administering to the subject a WT1 vaccine of the presentinvention, thereby reducing the incidence of relapse of an AML in asubject.

In another embodiment, the present invention provides a method ofbreaking a T cell tolerance of a subject to a WT1-expressing cancer, themethod comprising administering to the subject a WT1 vaccine of thepresent invention, thereby breaking a T cell tolerance to aWT1-expressing cancer.

In another embodiment, the present invention provides a method oftreating a subject having a WT1-expressing cancer, comprising (a)inducing in a donor formation and proliferation of human cytotoxic Tlymphocytes (CTL) that recognize a malignant cell of the cancer by amethod of the present invention; and (b) infusing the human CTL into thesubject, thereby treating a subject having a cancer.

In another embodiment, the present invention provides a method oftreating a subject having a WT 1-expressing cancer, comprising (a)inducing ex vivo formation and proliferation of human CTL that recognizea malignant cell of the cancer by a method of the present invention,wherein the human immune cells are obtained from a donor; and (b)infusing the human CTL into the subject, thereby treating a subjecthaving a cancer.

Methods for ex vivo immunotherapy are well known in the art and aredescribed, for example, in United States Patent Application SerialNumbers 2006/0057130, 2005/0221481, 2005/0214268, 2003/0175272,2002/0127718, and U.S. Pat. No. 5,229,115, which are incorporated hereinby reference. Additional methods are well known in the art and aredescribed, for example, in Davis I D et al (Blood dendritic cellsgenerated with Flt3 ligand and CD40 ligand prime CD8+ T cellsefficiently in cancer patients. J Immunother. 2006 September-October;29(5):499-511) and Mitchell M S et al (The cytotoxic T cell response topeptide analogs of the HLA-A*0201-restricted MUC1 signal sequenceepitope, M1.2. Cancer Immunol Immunother. 2006 JuI 28). Each methodrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofinducing the formation and proliferation of CTL specific for cells of aWT1-expressing cancer, the method comprising contacting a lymphocytepopulation with a vaccine of the present invention. In anotherembodiment, the vaccine is an APC associated with a peptide of thepresent invention. In another embodiment, the vaccine is an APCassociated with a mixture of peptides of the present invention. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, this invention provides a method of generating aheteroclitic immune response in a subject, wherein the heterocliticimmune response is directed against a WT1-expressing cancer, the methodcomprising administering to the subject a vaccine of the presentinvention, thereby generating a heteroclitic immune response.

In another embodiment, the present invention provides a method ofinducing an anti-mesothelioma immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising (a) a WT1 protein; or (b) a fragment of a WTprotein, thereby inducing an anti-mesothelioma immune response in asubject. In another embodiment, the mesothelioma is a malignantmesothelioma. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method ofinducing an anti-mesothelioma immune response in a subject, the methodcomprising the step of contacting the subject with an immunogeniccomposition comprising a nucleotide molecule encoding (a) a WT1 protein;or (b) a fragment of a WT1 protein, thereby inducing ananti-mesothelioma immune response in a subject. In another embodiment,the mesothelioma is a malignant mesothelioma. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method oftreating a subject with a mesothelioma, the method comprising the stepof administering to the subject an immunogenic composition comprising(a) a WT1 protein; or (b) a fragment of a WT protein, thereby treating asubject with a mesothelioma. In another embodiment, the mesothelioma isa malignant mesothelioma. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method oftreating a subject with a mesothelioma, the method comprising the stepof administering to the subject an immunogenic composition comprising anucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of aWT1 protein, thereby treating a subject with a mesothelioma. In anotherembodiment, the mesothelioma is a malignant mesothelioma. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofreducing an incidence of a mesothelioma, or its relapse, in a subject,the method comprising the step of administering to the subject animmunogenic composition comprising (a) a WT1 protein; or (b) a fragmentof a WT protein, thereby reducing an incidence of a mesothelioma, or itsrelapse, in a subject. In another embodiment, the mesothelioma is amalignant mesothelioma. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method ofreducing an incidence of a mesothelioma, or its relapse, in a subject,the method comprising the step of administering to the subject animmunogenic composition comprising a nucleotide molecule encoding (a) aWT1 protein; or (b) a fragment of a WT1 protein, thereby reducing anincidence of a mesothelioma, or its relapse, in a subject. In anotherembodiment, the mesothelioma is a malignant mesothelioma. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a target cell of an immune response elicited by amethod of the present invention presents the WT1 peptide of the presentinvention, or a corresponding WT1 fragment, on an HLA molecule. Inanother embodiment, the HLA molecule is an HLA class I molecule. Inother embodiments, the HLA molecule is any HLA class I subtype or HLAclass I molecule known in the art. In another embodiment, the immuneresponse against the WT1 peptide or fragment is a heteroclitic immuneresponse. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the WT1-expressing cancer is an acute myelogenousleukemia (AML). In another embodiment, the WT1-expressing cancer isassociated with a myelodysplastic syndrome (MDS). In another embodiment,the WT1-expressing cancer is an MDS. In another embodiment, theWT1-expressing cancer is a non-small cell lung cancer (NSCLC). Inanother embodiment, the WT1-expressing cancer is a Wilms' tumor. Inanother embodiment, the WT1-expressing cancer is a leukemia. In anotherembodiment, the WT1-expressing cancer is a hematological cancer. Inanother embodiment, the WT1-expressing cancer is a lymphoma. In anotherembodiment, the WT1-expressing cancer is a desmoplastic small round celltumor. In another embodiment, the WT1-expressing cancer is amesothelioma. In another embodiment, the WT1-expressing cancer is amalignant mesothelioma. In another embodiment, the WT1-expressing canceris a gastric cancer. In another embodiment, the WT1-expressing cancer isa colon cancer. In another embodiment, the WT1-expressing cancer is alung cancer. In another embodiment, the WT1-expressing cancer is abreast cancer. In another embodiment, the WT1-expressing cancer is agerm cell tumor. In another embodiment, the WT1-expressing cancer is anovarian cancer. In another embodiment, the WT 1-expressing cancer is auterine cancer. In another embodiment, the WT 1-expressing cancer is athyroid cancer. In another embodiment, the WT1-expressing cancer is ahepatocellular carcinoma. In another embodiment, the WT1-expressingcancer is a thyroid cancer. In another embodiment, the WT1-expressingcancer is a liver cancer. In another embodiment, the WT1-expressingcancer is a renal cancer. In another embodiment, the WT1-expressingcancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressingcancer is a sarcoma. In another embodiment, the WT1-expressing cancer isany other carcinoma or sarcoma.

In another embodiment, the WT1-expressing cancer is a solid tumor. Inanother embodiment, the solid tumor is associated with a WT1-expressingcancer. In another embodiment, the solid tumor is associated with amyelodysplastic syndrome (MDS). In another embodiment, the solid tumoris associated with a non-small cell lung cancer (NSCLC). In anotherembodiment, the solid tumor is associated with a lung cancer. In anotherembodiment, the solid tumor is associated with a breast cancer. Inanother embodiment, the solid tumor is associated with a colorectalcancer. In another embodiment, the solid tumor is associated with aprostate cancer. In another embodiment, the solid tumor is associatedwith an ovarian cancer. In another embodiment, the solid tumor isassociated with a renal cancer. In another embodiment, the solid tumoris associated with a pancreatic cancer. In another embodiment, the solidtumor is associated with a brain cancer. In another embodiment, thesolid tumor is associated with a gastrointestinal cancer. In anotherembodiment, the solid tumor is associated with a skin cancer. In anotherembodiment, the solid tumor is associated with a melanoma.

In another embodiment, a cancer or tumor treated by a method of thepresent invention is suspected to express WT1. In another embodiment,WT1 expression has not been verified by testing of the actual tumorsample. In another embodiment, the cancer or tumor is of a type known toexpress WT1 in many cases. In another embodiment, the type expresses WT1in the majority of cases.

Each type of WT1-expressing cancer or tumor, and cancer or tumorsuspected to express WT1, represents a separate embodiment of thepresent invention.

Any embodiments enumerated herein, regarding peptides, vaccines andcompositions of this invention can be employed in any of the methods ofthis invention, and each represents an embodiment thereof.

In another embodiment, multiple peptides of this invention are used tostimulate an immune response in methods of the present invention.

The methods disclosed herein will be understood by those in the art toenable design of other WT1-derived peptides. The methods further enabledesign of peptides binding to other HLA molecules. The methods furtherenable design of vaccines combining WT1-derived peptides of the presentinvention. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, vaccines of the present invention have theadvantage of activating or eliciting WT1-specific CD4<+> T cellscontaining a variety of different HLA class II alleles. In anotherembodiment, the vaccines have the advantage of activating or elicitingWT1-specific CD4<+> T cells in a substantial proportion of thepopulation (e.g. in different embodiments, 50%, 55%, 60%, 65%, 70%, 75%,80%. 85%, 90%, 95%, or greater than 95%). In another embodiment, thevaccines activate or elicit WT1-specific CD4<+> T cells in a substantialproportion of a particular population (e.g. American Caucasians). Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, methods of the present invention provide for animprovement in an immune response that has already been mounted by asubject. In another embodiment, methods of the present inventioncomprise administering the peptide, composition, or vaccine 2 or moretimes. In another embodiment, the peptides are varied in theircomposition, concentration, or a combination thereof. In anotherembodiment, the peptides provide for the initiation of an immuneresponse against an antigen of interest in a subject who has not yetinitiated an immune response against the antigen. In another embodiment,the CTL that are induced proliferate in response to presentation of thepeptide on the APC or cancer cell. In other embodiments, reference tomodulation of the immune response involves, either or both the humoraland cell-mediated arms of the immune system, which is accompanied by thepresence of Th2 and ThI T helper cells, respectively, or in anotherembodiment, each arm individually.

In other embodiments, the methods affecting the growth of a tumor resultin (1) the direct inhibition of tumor cell division, or (2) immune cellmediated tumor cell lysis, or both, which leads to a suppression in thenet expansion of tumor cells.

Inhibition of tumor growth by either of these two mechanisms can bereadily determined by one of ordinary skill in the art based upon anumber of well-known methods. In another embodiment, tumor inhibition isdetermined by measuring the actual tumor size over a period of time. Inanother embodiment, tumor inhibition can be determined by estimating thesize of a tumor (over a period of time) utilizing methods well known tothose of skill in the art. More specifically, a variety of radiologicimaging methods (e.g., single photon and positron emission computerizedtomography; see generally, “Nuclear Medicine in Clinical Oncology,”Winkler, C. (ed.) Springer-Verlag, New York, 1986), can be utilized toestimate tumor size. Such methods can also utilize a variety of imagingagents, including for example, conventional imaging agents (e.g.,Gallium-67 citrate), as well as specialized reagents for metaboliteimaging, receptor imaging, or immunologic imaging (e.g., radiolabeledmonoclonal antibody specific tumor markers). In addition,non-radioactive methods such as ultrasound (see, “UltrasonicDifferential Diagnosis of Tumors”, Kossoff and Fukuda, (eds.),Igaku-Shoin, New York, 1984), can also be utilized to estimate the sizeof a tumor.

In addition to the in vivo methods for determining tumor inhibitiondiscussed above, a variety of in vitro methods can be utilized in orderto predict in vivo tumor inhibition. Representative examples includelymphocyte mediated anti-tumor cytolytic activity determined forexample, by a <51>Cr release assay (Examples), tumor dependentlymphocyte proliferation (Ioannides, et al., J. Immunol.146(5):1700-1707, 1991), in vitro generation of tumor specificantibodies (Herlyn, et al., J. Immunol. Meth. 73:157-167, 1984), cell(e.g., CTL, helper T-cell) or humoral (e.g., antibody) mediatedinhibition of cell growth in vitro (Gazit, et al., Cancer ImmunolImmunother 35:135-144, 1992), and, for any of these assays,determination of cell precursor frequency (Vose, Int. J. Cancer30:135-142 (1982), and others.

In another embodiment, methods of suppressing tumor growth indicate agrowth state that is curtailed compared to growth without contact with,or exposure to a peptide of this invention. Tumor cell growth can beassessed by any means known in the art, including, but not limited to,measuring tumor size, determining whether tumor cells are proliferatingusing a <3>H-thymidine incorporation assay, or counting tumor cells.“Suppressing” tumor cell growth refers, in other embodiments, toslowing, delaying, or stopping tumor growth, or to tumor shrinkage. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment of methods and compositions of the presentinvention, WT1 expression is measured. In another embodiment, WT1transcript expression is measured. In another embodiment, WT1 proteinlevels in the tumor are measured. Each possibility represents a separateembodiment of the present invention.

Methods of determining the presence and magnitude of an immune responseare well known in the art. In another embodiment, lymphocyteproliferation assays, wherein T cell uptake of a radioactive substance,e.g. <3>H-thymidine is measured as a function of cell proliferation. Inother embodiments, detection of T cell proliferation is accomplished bymeasuring increases in interleukin-2 (IL-2) production, Ca<2+> flux, ordye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, CTL stimulation is determined by means known tothose skilled in the art, including, detection of cell proliferation,cytokine production and others. Analysis of the types and quantities ofcytokines secreted by T cells upon contacting ligand-pulsed targets canbe a measure of functional activity. Cytokines can be measured by ELISAor ELISPOT assays to determine the rate and total amount of cytokineproduction. (Fujihashi K. et al. (1993) J. Immunol. Meth. 160: 181;Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13:259).

In another embodiment, CTL activity is determined by <51>Cr-releaselysis assay. Lysis of peptide-pulsed <51>Cr-labeled targets byantigen-specific T cells can be compared for target cells pulsed withcontrol peptide. In another embodiment, T cells are stimulated with apeptide of this invention, and lysis of target cells expressing thenative peptide in the context of MHC can be determined. The kinetics oflysis as well as overall target lysis at a fixed timepoint (e.g., 4hours) are used, in another embodiment, to evaluate ligand performance.(Ware C. F. et al. (1983) J Immunol 131: 1312).

Methods of determining affinity of a peptide for an HLA molecule arewell known in the art. In another embodiment, affinity is determined byTAP stabilization assays.

In another embodiment, affinity is determined by competitionradioimmunoassay. In another embodiment, the following protocol isutilized: Target cells are washed two times in PBS with 1% bovine serumalbumin (BSA; Fisher Chemicals, Fairlawn, N.J.). Cells are resuspendedat 10<7>/ml on ice, and the native cell surface bound peptides arestripped for 2 minutes at 0 [deg.] C using citrate-phosphate buffer inthe presence of 3 mg/ml beta2 microglobulin. The pellet is resuspendedat 5×10<6> cells/ml in PBS/1% BSA in the presence of 3 mg/ml beta2microglobulin and 30 mg/ml deoxyribonuclease, and 200 ml aliquots areincubated in the presence or absence of HLA-specific peptides for 10 minat 20<0>C, then with <125>I-labeled peptide for 30 min at 2O<0>C. Totalbound <125>I is determined after two washes with PBS/2% BSA and one washwith PBS. Relative affinities are determined by comparison of escalatingconcentrations of the test peptide versus a known binding peptide.

In another embodiment, a specificity analysis of the binding of peptideto HLA on surface of live cells (e.g. SKLY-16 cells) is conducted toconfirm that the binding is to the appropriate HLA molecule and tocharacterize its restriction. This includes, in another embodiment,competition with excess unlabeled peptides known to bind to the same ordisparate HLA molecules and use of target cells which express the sameor disparate HLA types. This assay is performed, in another embodiment,on live fresh or 0.25% paraformaldehyde-fixed human PBMC, leukemia celllines and EBV-transformed T-cell lines of specific HLA types. Therelative avidity of the peptides found to bind MHC molecules on thespecific cells are assayed by competition assays as described aboveagainst <125>I-labeled peptides of known high affinity for the relevantHLA molecule, e.g., tyrosinase or HBV peptide sequence. [00165] Inanother embodiment, an HLA class II-binding peptide of methods andcompositions of the present invention is longer than the minimum lengthfor binding to an HLA class II molecule, which is, in anotherembodiment, about 12 AA. In another embodiment, increasing the length ofthe HLA class II-binding peptide enables binding to more than one HLAclass II molecule. In another embodiment, increasing the length enablesbinding to an HLA class II molecule whose binding motif is not known. Inanother embodiment, increasing the length enables binding to an HLAclass I molecule. In another embodiment, the binding motif of the HLAclass I molecule is known. In another embodiment, the binding motif ofthe HLA class I molecule is not known. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the peptides utilized in methods and compositionsof the present invention comprise a non-classical amino acid such as:1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991)J. Am Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine,(2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby (1991) TetrahedronLett. 32(41): 5769-5772); 2-aminotetrahydronaphthalene-2-carboxylic acid(Landis (1989) Ph.D. Thesis, University of Arizona);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.(1984) J. Takeda Res. Labs. 43:53-76) histidine isoquinoline carboxylicacid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138); andHIC (histidine cyclic urea), (Dharanipragada et al. (1993) Int. J. Pep.Protein Res. 42(1):68-77) and ((1992) Acta. Crst., Crystal Struc. Comm.48(IV): 1239-124).

In another embodiment, a peptide of this invention comprises an AAanalog or peptidomimetic, which, in other embodiments, induces or favorsspecific secondary structures. Such peptides comprise, in otherembodiments, the following: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a [beta]-turn inducingdipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838);[beta]-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett.29:5081-5082); [beta]-turn inducing analogs (Kemp et al. (1988)Tetrahedron Lett. 29:5057-5060); alpha-helix inducing analogs (Kemp etal. (1988) Tetrahedron Lett. 29:4935-4938); gamma-turn inducing analogs(Kemp et al. (1989) J. Org. Chem. 54:109:115); analogs provided by thefollowing references: Nagai and Sato (1985) Tetrahedron Lett.26:647-650; and DiMaio et al. (1989) J. Chem. Soc. Perkin Trans, p.1687; a GIy-Ala turn analog (Kahn et al. (1989) Tetrahedron Lett.30:2317); amide bond isostere (Jones et al. (1988) Tetrahedron Lett.29(31):3853-3856); tretrazol (Zabrocki et al. (1988) J. Am. Chem. Soc.110:5875-5880); DTC (Samanen et al. (1990) Int. J. Protein Pep. Res.35:501:509); and analogs taught in Olson et al. (1990) J. Am. Chem. Sci.112:323-333 and Garvey et al. (1990) J. Org. Chem. 55(3):936-940.Conformationally restricted mimetics of beta turns and beta bulges, andpeptides containing them, are described in U.S. Pat. No. 5,440,013,issued Aug. 8, 1995 to Kahn.

In other embodiments, a peptide of this invention is conjugated to oneof various other molecules, as described hereinbelow, which can be viacovalent or non-covalent linkage (complexed), the nature of whichvaries, in another embodiment, depending on the particular purpose. Inanother embodiment, the peptide is covalently or non-covalentlycomplexed to a macromolecular carrier, (e.g. an immunogenic carrier),including, but not limited to, natural and synthetic polymers, proteins,polysaccharides, polypeptides (amino acids), polyvinyl alcohol,polyvinyl pyrrolidone, and lipids. In another embodiment, a peptide ofthis invention is linked to a substrate. In another embodiment, thepeptide is conjugated to a fatty acid, for introduction into a liposome(U.S. Pat. No. 5,837,249). In another embodiment, a peptide of theinvention is complexed covalently or non-covalently with a solidsupport, a variety of which are known in the art. In another embodiment,linkage of the peptide to the carrier, substrate, fatty acid, or solidsupport serves to increase an elicited an immune response.

In other embodiments, the carrier is thyroglobulin, an albumin (e.g.human serum albumin), tetanus toxoid, polyamino acids such as poly(lysine:glutamic acid), an influenza protein, hepatitis B virus coreprotein, keyhole limpet hemocyanin, an albumin, or another carrierprotein or carrier peptide; hepatitis B virus recombinant vaccine, or anAPC. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the term “amino acid” (AA) refers to a naturalor, in another embodiment, an unnatural or synthetic AA, and caninclude, in other embodiments, glycine, D- or L optical isomers, AAanalogs, peptidomimetics, or combinations thereof.

In another embodiment, the terms “cancer,” “neoplasm,” “neoplastic” or“tumor,” are used interchangeably and refer to cells that have undergonea malignant transformation that makes them pathological to the hostorganism. Primary cancer cells (that is, cells obtained from near thesite of malignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.In another embodiment, a tumor is detectable on the basis of tumor mass;e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI),X-ray, ultrasound or palpation, and in another embodiment, is identifiedby biochemical or immunologic findings, the latter which is used toidentify cancerous cells, as well, in other embodiments.

Methods for synthesizing peptides are well known in the art. In anotherembodiment, the peptides of this invention are synthesized using anappropriate solid-state synthetic procedure (see for example, Stewardand Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco,Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23:451). The activity of these peptides is tested, in other embodiments,using assays as described herein.

In another embodiment, the peptides of this invention are purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for protein purification.In another embodiment, immuno-affinity chromatography is used, wherebyan epitope is isolated by binding it to an affinity column comprisingantibodies that were raised against that peptide, or a related peptideof the invention, and were affixed to a stationary support.

In another embodiment, affinity tags such as hexa-His (Invitrogen),Maltose binding domain (New England Biolabs), influenza coat sequence(Kolodziej et al. (1991) Meth. Enzymol. 194:508-509),glutathione-S-transferase, or others, are attached to the peptides ofthis invention to allow easy purification by passage over an appropriateaffinity column. Isolated peptides can also be physically characterized,in other embodiments, using such techniques as proteolysis, nuclearmagnetic resonance, and x-ray crystallography.

In another embodiment, the peptides of this invention are produced by invitro translation, through known techniques, as will be evident to oneskilled in the art. In another embodiment, the peptides aredifferentially modified during or after translation, e.g., byphosphorylation, glycosylation, cross-linking, acylation, proteolyticcleavage, linkage to an antibody molecule, membrane molecule or otherligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320).

In another embodiment, the peptides of this invention further comprise adetectable label, which in another embodiment, is fluorescent, or inanother embodiment, luminescent, or in another embodiment, radioactive,or in another embodiment, electron dense. In other embodiments, thedetectable label comprises, for example, green fluorescent protein(GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase(SEAP), beta-galactosidase, luciferase, <32>P, <125>I, <3>H and <14>C,fluorescein and its derivatives, rhodamine and its derivatives, dansyland umbelliferone, luciferin or any number of other such labels known toone skilled in the art. The particular label used will depend upon thetype of immunoassay used.

In another embodiment, a peptide of this invention is linked to asubstrate, which, in another embodiment, serves as a carrier. In anotherembodiment, linkage of the peptide to a substrate serves to increase anelicited an immune response.

In another embodiment, peptides of this invention are linked to othermolecules, as described herein, using conventional cross-linking agentssuch as carbodiimides. Examples of carbodiimides are1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC),1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.

In other embodiments, the cross-linking agents comprise cyanogenbromide, glutaraldehyde and succinic anhydride. In general, any of anumber of homo-bifunctional agents including a homo-bifunctionalaldehyde, a homo-bifunctional epoxide, a homo-bifunctional imido-ester,a homo-bifunctional N-hydroxysuccinimide ester, a homo-bifunctionalmaleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyldisulfide, a homo-bifunctional aryl halide, a homo-bifunctionalhydrazide, a homo-bifunctional diazonium derivative and ahomo-bifunctional photoreactive compound can be used. Also envisioned,in other embodiments, are hetero-bifunctional compounds, for example,compounds having an amine-reactive and a sulfhydryl-reactive group,compounds with an amine-reactive and a photoreactive group and compoundswith a carbonyl-reactive and a sulfhydryl-reactive group.

In other embodiments, the homo-bifunctional cross-linking agents includethe bifunctional N-hydroxysuccinimide estersdithiobis(succinimidylpropionate), disuccinimidyl suberate, anddisuccinimidyl tartarate; the bifunctional imido-esters dimethyladipimidate, dimethyl pimelimidate, and dimethyl suberimidate; thebifunctional sulfhydryl-reactive crosslinkers1,4-di-[3′-(2′-pyridyldithio)propionamido]butane, bismaleimidohexane,and bis-N-maleimido-1,8-octane; the bifunctional aryl halides1,5-difluoro-2,4-dinitrobenzene and4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactiveagents such as bis-[b-(4-azidosalicylamido)ethyl]disulfide; thebifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde,glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipicacid dihydrazide, carbohydrazide, and succinic acid dihydrazide; thebifunctional diazoniums o-tolidine, diazotized and bis-diazotizedbenzidine; the bifunctional alkylhalidesN1N′-ethylene-bis(iodoacetamide), N1N′-hexamethylene-bis(iodoacetamide),N1N′-undecamethylene-bis(iodoacetamide), as well as benzylhalides andhalomustards, such as ala′-diiodo-p-xylene sulfonic acid andtri(2-chloroethyl)amine, respectively,

In other embodiments, hetero-bifunctional cross-linking agents used tolink the peptides to other molecules, as described herein, include, butare not limited to, SMCC(succinimidyl-4-(N-rnaleimidomethyl)cyclohexane-1-carboxylate), MB S(m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB(N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB(succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS(N-(.gamma.-maleimidobutyryloxy)succmimide ester), MPBH(4-(4-N-maleimidophenyl) butyric acid hydrazide), M2C2H(4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide), SMPT(succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), and SPDP(N-succinimidyl 3-(2-pyridyldithio)propionate).

In another embodiment, the peptides of the invention are formulated asnon-covalent attachment of monomers through ionic, adsorptive, orbiospecific interactions. Complexes of peptides with highly positivelyor negatively charged molecules can be accomplished, in anotherembodiment, through salt bridge formation under low ionic strengthenvironments, such as in deionized water. Large complexes can becreated, in another embodiment, using charged polymers such aspoly-(L-glutamic acid) or poly-(L-lysine), which contain numerousnegative and positive charges, respectively. In another embodiment,peptides are adsorbed to surfaces such as microparticle latex beads orto other hydrophobic polymers, forming non-covalently associatedpeptide-superantigen complexes effectively mimicking cross-linked orchemically polymerized protein, in other embodiments. In anotherembodiment, peptides are non-covalently linked through the use ofbiospecific interactions between other molecules. For instance,utilization of the strong affinity of biotin for proteins such as avidinor streptavidin or their derivatives could be used to form peptidecomplexes. The peptides, according to this aspect, and in anotherembodiment, can be modified to possess biotin groups using commonbiotinylation reagents such as the N-hydroxysuccinimidyl ester ofD-biotin (NHS-biotin), which reacts with available amine groups.

In another embodiment, a peptide of the present invention is linked to acarrier. In another embodiment, the carrier is KLH. In otherembodiments, the carrier is any other carrier known in the art,including, for example, thyroglobulin, albumins such as human serumalbumin, tetanus toxoid, polyamino acids such as poly (lysine:glutamicacid), influenza, hepatitis B virus core protein, hepatitis B virusrecombinant vaccine and the like. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the peptides of this invention are conjugated toa lipid, such as P3 CSS. In another embodiment, the peptides of thisinvention are conjugated to a bead.

In another embodiment, the compositions of this invention furthercomprise immunomodulating compounds. In other embodiments, theimmunomodulating compound is a cytokine, chemokine, or complementcomponent that enhances expression of immune system accessory oradhesion molecules, their receptors, or combinations thereof. In someembodiments, the immunomodulating compound include interleukins, forexample interleukins 1 to 15, interferons alpha, beta or gamma, tumournecrosis factor, granulocyte-macrophage colony stimulating factor(GM-CSF), macrophage colony stimulating factor (M-CSF), granulocytecolony stimulating factor (G-CSF), chemokines such as neutrophilactivating protein (NAP), macrophage chemoattractant and activatingfactor (MCAF), RANTES, macrophage inflammatory peptides MIP-Ia andMIP-Ib, complement components, or combinations thereof. In otherembodiments, the immunomodulating compound stimulate expression, orenhanced expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L,B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors, or combinationthereof.

In another embodiment, the immunomodulatory compound induces or enhancesexpression of co-stimulatory molecules that participate in the immuneresponse, which include, in some embodiments, CD40 or its ligand, CD28,CTLA-4 or a B7 molecule. In another embodiment, the immunomodulatorycompound induces or enhances expression of a heat stable antigen (HSA)(Liu Y. et al. (1992) J. Exp. Med. 175:437-445), chondroitinsulfate-modified MHC invariant chain (Ii-CS) (Naujokas M. F. et al(1993) Cell 74:257-268), or an intracellular adhesion molecule 1(ICAM-I) (Van R. H. (1992) Cell 71: 1065-1068), which assists, inanother embodiment, co-stimulation by interacting with their cognateligands on the T cells.

In another embodiment, the composition comprises a solvent, includingwater, dispersion media, cell culture media, isotonic agents and thelike. In another embodiment, the solvent is an aqueous isotonic bufferedsolution with a pH of around 7.0. In another embodiment, the compositioncomprises a diluent such as water, phosphate buffered saline, or saline.In another embodiment, the composition comprises a solvent, which isnon-aqueous, such as propyl ethylene glycol, polyethylene glycol andvegetable oils.

In another embodiment, the composition is formulated for administrationby any of the many techniques known to those of skill in the art. Forexample, this invention provides for administration of thepharmaceutical composition parenterally, intravenously, subcutaneously,intradermally, intramucosally, topically, orally, or by inhalation.

In another embodiment, the vaccine comprising a peptide of thisinvention further comprises a cell population, which, in anotherembodiment, comprises lymphocytes, monocytes, macrophages, dendriticcells, endothelial cells, stem cells or combinations thereof, which, inanother embodiment are autologous, syngeneic or allogeneic, with respectto each other. In another embodiment, the cell population comprises apeptide of the present invention. In another embodiment, the cellpopulation takes up the peptide. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the cell populations of this invention areobtained from in vivo sources, such as, for example, peripheral blood,leukopheresis blood product, apheresis blood product, peripheral lymphnodes, gut associated lymphoid tissue, spleen, thymus, cord blood,mesenteric lymph nodes, liver, sites of immunologic lesions, e.g.synovial fluid, pancreas, cerebrospinal fluid, tumor samples,granulomatous tissue, or any other source where such cells can beobtained. In another embodiment, the cell populations are obtained fromhuman sources, which are, in other embodiments, from human fetal,neonatal, child, or adult sources. In another embodiment, the cellpopulations of this invention are obtained from animal sources, such as,for example, porcine or simian, or any other animal of interest. Inanother embodiment, the cell populations of this invention are obtainedfrom subjects that are normal, or in another embodiment, diseased, or inanother embodiment, susceptible to a disease of interest.

In another embodiment, the cell populations of this invention areseparated via affinity-based separation methods. Techniques for affinityseparation include, in other embodiments, magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents joined to a monoclonal antibody or use in conjunction with amonoclonal antibody, for example, complement and cytotoxins, and“panning” with an antibody attached to a solid matrix, such as a plate,or any other convenient technique. In other embodiment, separationtechniques include the use of fluorescence activated cell sorters, whichcan have varying degrees of sophistication, such as multiple colorchannels, low angle and obtuse light scattering detecting channels,impedance channels, etc. In other embodiments, any technique thatenables separation of the cell populations of this invention can beemployed, and is to be considered as part of this invention.

In another embodiment, the dendritic cells are from the diversepopulation of morphologically similar cell types found in a variety oflymphoid and non-lymphoid tissues, qualified as such (Steinman (1991)Ann. Rev. Immunol. 9:271-296). In another embodiment, the dendriticcells used in this invention are isolated from bone marrow, or inanother embodiment, derived from bone marrow progenitor cells, or, inanother embodiment, from isolated from/derived from peripheral blood, orin another embodiment, derived from, or are a cell line.

In another embodiment, the cell populations described herein areisolated from the white blood cell fraction of a mammal, such as amurine, simian or a human (See, e.g., WO 96/23060). The white blood cellfraction can be, in another embodiment, isolated from the peripheralblood of the mammal.

Methods of isolating dendritic cells are well known in the art. Inanother embodiment, the DC are isolated via a method which includes thefollowing steps: (a) providing a white blood cell fraction obtained froma mammalian source by methods known in the art such as leukophoresis;(b) separating the white blood cell fraction of step (a) into four ormore subfractions by countercurrent centrifugal elutriation; (c)stimulating conversion of monocytes in one or more fractions from step(b) to dendritic cells by contacting the cells with calcium ionophore,GM-CSF and IL-13 or GM-CSF and IL-4, (d) identifying the dendriticcell-enriched fraction from step (c); and (e) collecting the enrichedfraction of step (d), preferably at about 4[deg.] C.

In another embodiment, the dendritic cell-enriched fraction isidentified by fluorescence-activated cell sorting, which identifies atleast one of the following markers: HLA-DR, HLA-DQ, or B7.2, and thesimultaneous absence of the following markers: CD3, CD14, CD16, 56, 57,and CD 19, 20.

In another embodiment, the cell population comprises lymphocytes, whichare, in another embodiment, T cells, or in another embodiment, B cells.The T cells are, in other embodiments, characterized as NK cells, helperT cells, cytotoxic T lymphocytes (CTL), TBLs, naive T cells, orcombinations thereof. It is to be understood that T cells which areprimary, or cell lines, clones, etc. are to be considered as part ofthis invention. In another embodiment, the T cells are CTL, or CTLlines, CTL clones, or CTLs isolated from tumor, inflammatory, or otherinfiltrates.

In another embodiment, hematopoietic stem or early progenitor cellscomprise the cell populations used in this invention. In anotherembodiment, such populations are isolated or derived, by leukaphoresis.In another embodiment, the leukapheresis follows cytokineadministration, from bone marrow, peripheral blood (PB) or neonatalumbilical cord blood. In another embodiment, the stem or progenitorcells are characterized by their surface expression of the surfaceantigen marker known as CD34<+>, and exclusion of expression of thesurface lineage antigen markers, Lin-.

In another embodiment, the subject is administered a peptide,composition or vaccine of this invention, in conjunction with bonemarrow cells. In another embodiment, the administration together withbone marrow cells embodiment follows previous irradiation of thesubject, as part of the course of therapy, in order to suppress, inhibitor treat cancer in the subject.

In another embodiment, the phrase “contacting a cell” or “contacting apopulation” refers to a method of exposure, which can be, in otherembodiments, direct or indirect. In another embodiment, such contactcomprises direct injection of the cell through any means well known inthe art, such as microinjection. It is also envisaged, in anotherembodiment, that supply to the cell is indirect, such as via provisionin a culture medium that surrounds the cell, or administration to asubject, via any route well known in the art, and as described herein.

In another embodiment, CTL generation of methods of the presentinvention is accomplished in vivo, and is effected by introducing into asubject an antigen presenting cell contacted in vitro with a peptide ofthis invention (See for example Paglia et al. (1996) J. Exp. Med.183:317-322).

In another embodiment, the peptides of methods and compositions of thepresent invention are delivered to APC. In another embodiment, thepeptide-pulsed APC are administered to a subject to elicit and immuneresponse or treat or inhibit growth or recurrence of a tumor. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the peptides are delivered to APC in the form ofcDNA encoding the peptides. In another embodiment, the term“antigen-presenting cells” (APC) refers to dendritic cells (DC),monocytes/macrophages, B lymphocytes or other cell type(s) expressingthe necessary MHC/co-stimulatory molecules, which effectively allow forT cell recognition of the presented peptide. In another embodiment, theAPC is a cancer cell. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the CTL are contacted with 2 or more APCpopulations. In another embodiment, the 2 or more APC populationspresent different peptides. Each possibility represents a separateembodiment of the present invention.

In another embodiment, techniques that lead to the expression of antigenin the cytosol of APC (e.g. DC) are used to deliver the peptides to theAPC. Methods for expressing antigens on APC are well known in the art.In another embodiment, the techniques include (1) the introduction intothe APC of naked DNA encoding a peptide of this invention, (2) infectionof APC with recombinant vectors expressing a peptide of this invention,and (3) introduction of a peptide of this invention into the cytosol ofan APC using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med.184:465-472; Rouse et al. (1994) J. Virol. 68:5685-5689; and Nair et al.(1992) J. Exp. Med. 175:609-612).

In another embodiment, foster APC such as those derived from the humancell line 174xCEM.T2, referred to as T2, which contains a mutation inits antigen processing pathway that restricts the association ofendogenous peptides with cell surface MHC class I molecules (Zweerink etal. (1993) J. Immunol. 150:1763-1771), are used, as exemplified herein.

In another embodiment, as described herein, the subject is exposed to apeptide, or a composition/cell population comprising a peptide of thisinvention, which differs from the native protein expressed, whereinsubsequently a host immune cross-reactive with the nativeprotein/antigen develops.

In another embodiment, the subject, as referred to in any of the methodsor embodiments of this invention is a human. In other embodiments, thesubject is a mammal, which can be a mouse, rat, rabbit, hamster, guineapig, horse, cow, sheep, goat, pig, cat, dog, monkey, or ape. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, peptides, vaccines, and compositions of thisinvention stimulate an immune response that results in tumor cell lysis.

In another embodiment, any of the methods described herein is used toelicit CTL, which are elicited in vitro. In another embodiment, the CTLare elicited ex-vivo. In another embodiment, the CTL are elicited invitro. The resulting CTL, are, in another embodiment, administered tothe subject, thereby treating the condition associated with the peptide,an expression product comprising the peptide, or a homologue thereof.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the method entails introduction of the geneticsequence that encodes the peptides of this invention using, e.g., one ormore nucleic acid delivery techniques. Nucleic acids of the inventioninclude, in another embodiment, DNA, RNA and mixtures of DNA and RNA,alone or in conjunction with non-nucleic acid components. In anotherembodiment, the method comprises administering to the subject a vectorcomprising a nucleotide sequence, which encodes a peptide of the presentinvention (Tindle, R. W. et al. Virology (1994) 200:54). In anotherembodiment, the method comprises administering to the subject naked DNAwhich encodes a peptide, or in another embodiment, two or more peptidesof this invention (Nabel, et al. PNAS-USA (1990) 90: 11307). In anotherembodiment, multi-epitope, analogue-based cancer vaccines are utilized(Fikes et al, Design of multi-epitope, analogue-based cancer vaccines.Expert Opin Biol Ther. 2003 September; 3(6):985-93). Each possibilityrepresents a separate embodiment of the present invention.

Nucleic acids can be administered to a subject via any means as is knownin the art, including parenteral or intravenous administration, or inanother embodiment, by means of a gene gun. In another embodiment, thenucleic acids are administered in a composition, which correspond, inother embodiments, to any embodiment listed herein.

Vectors for use according to methods of this invention can comprise anyvector that facilitates or allows for the expression of a peptide ofthis invention. Vectors comprises, in some embodiments, attenuatedviruses, such as vaccinia or fowlpox, such as described in, e.g., U.S.Pat. No. 4,722,848, incorporated herein by reference. In anotherembodiment, the vector is BCG (Bacille Calmette Guerin), such asdescribed in Stover et al. (Nature 351:456-460 (1991)). A wide varietyof other vectors useful for therapeutic administration or immunizationof the peptides of the invention, e.g., Salmonella typhi vectors and thelike, will be apparent to those skilled in the art from the descriptionherein.

In another embodiment, the vector further encodes for animmunomodulatory compound, as described herein. In another embodiment,the subject is administered an additional vector encoding same,concurrent, prior to or following administration of the vector encodinga peptide of this invention to the subject.

In another embodiment, the peptides, compositions and vaccines of thisinvention are administered to a subject, or utilized in the methods ofthis invention, in combination with other anticancer compounds andchemotherapeutics, including monoclonal antibodies directed againstalternate cancer antigens, or, in another embodiment, epitopes thatconsist of an AA sequence which corresponds to, or in part to, that fromwhich the peptides of this invention are derived.

Various embodiments of dosage ranges are contemplated by this invention.[mu] refers to micro; [mu]g referring to microgram or micrograms. Inanother embodiment, the dosage is 20 [mu]g per peptide per day. Inanother embodiment, the dosage is 10 [mu]g/peptide/day. In anotherembodiment, the dosage is 30 [mu]g/peptide/day. In another embodiment,the dosage is 40 [mu]g/peptide/day. In another embodiment, the dosage is60 [mu]g/peptide/day. In another embodiment, the dosage is 80[mu]g/peptide/day. In another embodiment, the dosage is 100[mu]g/peptide/day. In another embodiment, the dosage is 150[mu]g/peptide/day. In another embodiment, the dosage is 200[mu]g/peptide/day. In another embodiment, the dosage is 300[mu]g/peptide/day. In another embodiment, the dosage is 400[mu]g/peptide/day. In another embodiment, the dosage is 600[mu]g/peptide/day. In another embodiment, the dosage is 800[mu]g/peptide/day. In another embodiment, the dosage is 1000[mu]g/peptide/day. In another embodiment, the dosage is 1500[mu]g/peptide/day. In another embodiment, the dosage is 2000[mu]g/peptide/day.

In another embodiment, the dosage is 10 [mu]g/peptide/dose. In anotherembodiment, the dosage is 30 [mu]g/peptide/dose. In another embodiment,the dosage is 40 [mu]g/peptide/dose. In another embodiment, the dosageis 60 [mu]g/peptide/dose. In another embodiment, the dosage is 80[mu]g/peptide/dose. In another embodiment, the dosage is 100[mu]g/peptide/dose. In another embodiment, the dosage is 150[mu]g/peptide/dose. In another embodiment, the dosage is 200[mu]g/peptide/dose. In another embodiment, the dosage is 300[mu]g/peptide/dose. In another embodiment, the dosage is 400[mu]g/peptide/dose. In another embodiment, the dosage is 600[mu]g/peptide/dose. In another embodiment, the dosage is 800[mu]g/peptide/dose. In another embodiment, the dosage is 1000[mu]g/peptide/dose. In another embodiment, the dosage is 1500[mu]g/peptide/dose. In another embodiment, the dosage is 2000[mu]g/peptide/dose.

In another embodiment, the dosage is 10-20 [mu]g/peptide/dose. Inanother embodiment, the dosage is 20-30 [mu]g/peptide/dose. In anotherembodiment, the dosage is 20-40 [mu]g/peptide/dose. In anotherembodiment, the dosage is 30-60 [mu]g/peptide/dose. In anotherembodiment, the dosage is 40-80 [mu]g/peptide/dose. In anotherembodiment, the dosage is 50-100 [mu]g/peptide/dose. In anotherembodiment, the dosage is 50-150 [mu]g/peptide/dose. In anotherembodiment, the dosage is 100-200 [mu]g/peptide/dose. In anotherembodiment, the dosage is 200-300 [mu]g/peptide/dose. In anotherembodiment, the dosage is 300-400 [mu]g/peptide/dose. In anotherembodiment, the dosage is 400-600 [mu]g/peptide/dose. In anotherembodiment, the dosage is 500-800 [mu]g/peptide/dose. In anotherembodiment, the dosage is 800-1000 [mu]g/peptide/dose. In anotherembodiment, the dosage is 1000-1500 [mu]g/peptide/dose. In anotherembodiment, the dosage is 1500-2000 [mu]g/peptide/dose.

In another embodiment, the total amount of peptide per dose or per dayis one of the above amounts. In another embodiment, the total peptidedose per dose is one of the above amounts.

Each of the above doses represents a separate embodiment of the presentinvention.

Various embodiments of dosage ranges are contemplated by this invention.In another embodiment, the dosage is 20 mg per peptide per day. Inanother embodiment, the dosage is 10 mg/peptide/day. In anotherembodiment, the dosage is 30 mg/peptide/day. In another embodiment, thedosage is 40 mg/peptide/day. In another embodiment, the dosage is 60mg/peptide/day. In another embodiment, the dosage is 80 mg/peptide/day.In another embodiment, the dosage is 100 mg/peptide/day. In anotherembodiment, the dosage is 150 mg/peptide/day. In another embodiment, thedosage is 200 mg/peptide/day. In another embodiment, the dosage is 300mg/peptide/day. In another embodiment, the dosage is 400 mg/peptide/day.In another embodiment, the dosage is 600 mg/peptide/day. In anotherembodiment, the dosage is 800 mg/peptide/day. In another embodiment, thedosage is 1000 mg/peptide/day.

In another embodiment, the dosage is 10 mg/peptide/dose. In anotherembodiment, the dosage is 30 mg/peptide/dose. In another embodiment, thedosage is 40 mg/peptide/dose. In another embodiment, the dosage is 60mg/peptide/dose. In another embodiment, the dosage is 80mg/peptide/dose. In another embodiment, the dosage is 100mg/peptide/dose. In another embodiment, the dosage is 150mg/peptide/dose. In another embodiment, the dosage is 200mg/peptide/dose. In another embodiment, the dosage is 300mg/peptide/dose. In another embodiment, the dosage is 400mg/peptide/dose. In another embodiment, the dosage is 600mg/peptide/dose. In another embodiment, the dosage is 800mg/peptide/dose. In another embodiment, the dosage is 1000mg/peptide/dose.

In another embodiment, the dosage is 10-20 mg/peptide/dose. In anotherembodiment, the dosage is 20-30 mg/peptide/dose. In another embodiment,the dosage is 20-40 mg/peptide/dose. In another embodiment, the dosageis 30-60 mg/peptide/dose. In another embodiment, the dosage is 40-80mg/peptide/dose. In another embodiment, the dosage is 50-100mg/peptide/dose. In another embodiment, the dosage is 50-150mg/peptide/dose. In another embodiment, the dosage is 100-200mg/peptide/dose. In another embodiment, the dosage is 200-300mg/peptide/dose. In another embodiment, the dosage is 300-400mg/peptide/dose. In another embodiment, the dosage is 400-600mg/peptide/dose. In another embodiment, the dosage is 500-800mg/peptide/dose. In another embodiment, the dosage is 800-1000mg/peptide/dose.

In another embodiment, the total amount of peptide per dose or per dayis one of the above amounts. In another embodiment, the total peptidedose per dose is one of the above amounts.

Each of the above doses represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a kit comprising apeptide, composition or vaccine of the present invention. In anotherembodiment, the kit further comprises a label or packaging insert. Inanother embodiment, the kit is used for detecting a WT1-specific CD4response through the use of a delayed-type hypersensitivity test. Inanother embodiment, the kit is used for any other method enumeratedherein. In another embodiment, the kit is used for any other methodknown in the art. Each possibility represents a separate embodiment ofthe present invention.

Example 1 Materials and Methods

Peptide Design.

Using three computer-based predictive algorisms BIMAS(http://www-bimas.cit.nih.gov/cgi-bin/molbio/ken_parker_comboform),SYFPEITHI (http://www.syfpeithi.de/) and RANKPEP(http://bio.dfci.harvard.edu/Tools/rankpep.html), epitopes were selectedfor both CD8 and CD4 T cells by starting with the native WT1 proteinsequences that are capable of inducing immune response in normal donors.Heteroclitic peptides were designed by altering a single amino acid inthe anchor residues of the native peptides for class I, which resultedin a higher predicted binding than its native sequences. The class IIpeptides were designed by adding flanking residues to the class Ipeptides, in order to simultaneously stimulate both CD4 and CD8 T cells.While many sequences can be predicted by the algorithms, these models donot predict binding to MHC when tested on live cells in 30% of cases(Gomez-Nunez et al. Leuk Res. 2006; 30(10): 1293-8), therefore in vitrotesting is necessary. In addition, even if binding is demonstrated, acytotoxic T cell response may not occur, requiring additional in vitrostudy.

Peptide Synthesis.

All peptides were purchased and synthesized by Genemed Synthesis, Inc.(San Antonio, Tex.). Peptides were sterile with purity of 70% to 90%.The peptides were dissolved in DMSO and diluted in saline at 5 mg/mL andstored at −80° C. Control peptides used are: for HLA-DR.B1:JAK-2-derived DR.B1-binding peptide JAK2-DR (GVCVCGDENILVQEF; SEQ IDNO:59) or BCR.ABL-derived peptide (IVHSATGFKQSSKALQRPVASDFEP; SEQ IDNO:60); for HLA-A0201: ewing sarcoma-derived peptide EW (QLQNPSYDK; SEQID NO:61) and for HLA-A2402: prostate-specific membrane antigen(PMSA)-derived peptide 624-632 (TYSVSFDSL; SEQ ID NO:62).

Cells Lines, Cytokines and Antibodies.

Human leukemia cell lines BA25 and HL-60 were used as a targets formeasuring cytotoxicity of T cells. Human granulocyte-macrophagecolony-stimulating factor (GM-CSF), interleukin (IL)-1beta, IL-4, IL-6,IL-15, tumor necrosis factor (TNF)-alpha and prostaglandin E2 (PGE2)were purchased from R&D Systems (Minneapolis, Minn.). Beta2-microglobulin (b2-m) was purchased from Sigma (St. Louis, Mo.). Theantibodies used for immunofluorescence assays including mAbs to humanCD3, CD4, CD8, HLA-A2 (clone BB7.2) and isotype controls were obtainedfrom BD Biosciences (San Diego, Calif.). Cell isolation kits for CD14and CD3 were purchased from Miltenyi Biotec. (Bergisch Gladbach,Germany).

T2 Assay for Peptide Binding.

T2 cells (TAP-, HLA-A0201+) were incubated overnight at 37° C. at 1×10⁶cells/ml in FCS-free RPMI medium supplemented with 10 ug/ml humanbeta-2m (Sigma, St Louis, Mo., USA) in the absence (negative control) orpresence peptides at various final concentrations (50, 10 and 2 ug/ml).Brefeldin A (Sigma) at 5 ug/ml was added to the cultures for the finaltwo hrs of incubation. Then T2 cells were washed and stained withanti-HLA-A2.1 (BB7.2) mAb conjugated to FITC for 30 min at 4° C. andfollowed by washing with staining buffer (PBS plus 1% FBS and 0.02%azide). The expression of the HLA-A2 on the cell surface was measured byflow cytometry on a FACScalibur (Becton Dickinson) and analyzed withFlowJo 9.6.3 software.

In Vitro Stimulation and Human T-Cell Cultures.

Peripheral blood mononuclear cells (PBMCs) from HLA-typed healthy donorswere obtained by Ficoll density centrifugation. CD14+ monocytes wereisolated by positive selection using mAb to human CD14 coupled withmagnetic beads (Miltenyi Biotec) and were used for the first stimulationof T cells. The CD14− fraction of PBMC were used for isolation of CD3,by negative immunomagnetic cell separation using a pan T cell isolationkit (Miltenyi Biotec). The purity of the cells was always more than 98%.T cells were stimulated for 7 days in the presence of RPMI 1640supplemented with 5% autologous plasma (AP), 20 ug/mL syntheticpeptides, 1 ug/mL B2-m, and 10 ng/mL IL-15. Monocyte-derived dendriticcells (DCs) were generated from CD14+ cells, by culturing the cells inRPMI 1640 medium supplemented with 1% AP, 500 units/mL recombinant IL-4,and 1,000 units/mL GM-CSF. On days 2 and 4 of incubation, fresh mediumwith IL-4 and GM-CSF was either added or replaced half of the culturemedium. On day 5, 20 ug/mL class II peptide was added to the immatureDCs, for the processing. On day 6, maturation cytokine cocktail wasadded (Dao et al. Plos One 2009; 4(8):e6730). On day 7 or 8, T cellswere re-stimulated with mature DCs, with IL-15. In most cases, T cellswere stimulated 3 times in the same manner, using either DCs or CD14+cells as antigen-presenting cells (APCs). A week after finalstimulation, the peptide-specific T cell response was examined by IFN-genzyme-linked immunospot (ELISPOT) assay and the cytotoxicity wastested, by ⁵¹chromium (Cr)-release assay.

IFN-g ELISPOT.

HA-Multiscreen plates (Millipore) were coated with 100 uL of mouseanti-human IFN-g antibody (10 Ag/mL; clone 1-D1K; Mabtech) in PBS,incubated overnight at 4 C, washed with PBS to remove unbound antibody,and blocked with RPMI 1640/10% autologous plasma (AP) for 2 h at 37° C.Purified CD3+ T cells (>98% pure) were plated with either autologousCD14+ (10:1 E: APC ratio) or autologous DCs (30:1 E: APC ratio). Varioustest peptides were added to the wells at 20 ug/mL. Negative controlwells contained APCs and T cells without peptides or with irrelevantpeptides. Positive control wells contained T cells plus APCs plus 20ug/mL phytohemagglutinin (PHA, Sigma). All conditions were done intriplicates. Microtiter plates were incubated for 20 h at 37° C. andthen extensively washed with PBS/0.05% Tween and 100 ul/wellbiotinylated detection antibody against human IFN-g (2 ug/mL; clone7-B6-1; Mabtech) was added. Plates were incubated for an additional 2 hat 37° C. and spot development was done as described (Dao et al., op.cit.). Spot numbers were automatically determined with the use of acomputer-assisted video image analyzer with KS ELISPOT 4.0 software(Carl Zeiss Vision).

⁵¹Chromium Release Assay.

The presence of specific CTLs was measured in a standard chromiumrelease assay as described (Dao et al., op. cit.). Briefly, target cellsalone, or pulsed with 50 ug/mL of synthetic peptides for 2 hours (insome cases for over night) at 37° C., are labeled with 50 uCi/millioncells of Na₂ ⁵¹CrO₄ (NEN Life Science Products, Inc.). After extensivewashing, target cells are incubated with T cells at E:T ratios rangingfrom 100:1 to 10:1. All conditions were done in triplicate. Plates wereincubated for 4-5 hrs at 37° C. in 5% CO2. Supernatant fluids wereharvested and radioactivity was measured in a gamma counter. Percentagespecific lysis was determined from the following formula: [(experimentalrelease−spontaneous release)/(maximum release−spontaneousrelease)]×100%. Maximum release was determined by lysis of radiolabeledtargets in 1% SDS.

Example 2 Binding of the Native and its Analogue Peptides to HLA-A0201and HLA-A2402

Using a pool of 15 mer overlapping peptides spanning human WT1 proteinto sensitize human T cells in vitro, the sequence 239-248 (NQMNLGATL;SEQ ID NO:5; herein abbreviated NQM or) has recently been identified asan immunogenic CD8 T cell epitope in the context of HLA-A2402(Doubrovina et al., Blood 2012; 123(8):1633-46). In order to generateanalog peptides with stronger immunogenicity, the prediction scores ofthe native peptide and possible analogs with various amino acidsubstitutions in the position 2 and 9 (class I anchor residues) wasscreened, using three online available databases (BIMAS, RANKPEP andSYFPEITHI). The predicted binding scores from all three databases showedbetter binding of the native NQMNLGATL (SEQ ID NO:5) peptide toHLA-A0201 than HLA-A2402 molecule (Table I). When the glutamine at theposition 2 was substituted by leucine, the binding score to HLA-A2402remained at the similar level by all 3 prediction programs. However, asignificantly stronger binding score was predicted for HLA-A0201. On theother hand, when the glutamine at the position 2 was substituted bytyrosine, binding score to HLA-A2402 was dramatically improved, showingabout 90-fold increased binding by BIMAS prediction. All three peptideswere predicted to be cleaved at c-terminal by RANKPEP algorithm,suggesting the processing of the peptide fragment. The binding score waschecked by substitution with various amino acids at position 9 but noneof them showed a significant improved binding compared to thesubstitution at the position 2. Therefore, the two analogue peptidesNLMNLGATL (SEQ ID NO:6; herein abbreviated NLM or A24-het-1) andNYMNLGATL (SEQ ID NO:7; herein abbreviated NYM or A24-het-2) wereselected for further studies.

TABLE 1 Predictive binding scores of the peptides to HLA-A0201 and A2402Sequences BIMAS SYFPETHI RANKPEP (score; % opt) (p 239-247) HLA-A0201HLA-A2402 HLA-A0201 HLA-A24 HLA-A0201 HLA-A2402 NQMNLGATL 8.014 7.200 1710 34; 26.56% 10.482; (SEQ ID NO: 5) Cleaved 27.23%, Cleaved NLMNLGATL79.041 7.2 26 10 78; 60.94% 8.948; (SEQ ID NO: 6) Cleaved 23.24%,Cleaved NYMNLGATL 0.011 360.000  9 20 41; 32.03% 23.573; (SEQ ID NO: 7)Cleaved 61.22%, Cleaved

Example 3 Binding of the Peptides to HLA-A0201 and HLA-A2402 Molecules

The immunogenicity of MHC class I-restricted peptides requires thecapacity to bind and stabilize MHC class I molecules on the live cellsurface. Moreover, the computer prediction has only up to 70% accuracy;therefore, direct measurement was sought of the strength of theinteraction between the peptides and the HLA-A0201 molecules using aconventional binding and stabilization assay that uses theantigen-transporting-deficient (TAP2 negative) HLA-A0201 human T2 cells.T2 cells lack TAP function and consequently are defective in properlyloading class I molecules with antigenic peptides generated in thecytosol. The association of exogenously added peptides withthermolabile, empty HLA-A0201 molecules stabilizes them and results inan increase in the level of surface HLA-A0201 recognizable by specificanti-HLA-A0201 mAb such as BB7.2.

The T2 binding assay showed that native NQMNLGATL (SEQ ID NO:5) peptidedid not increase the HLA-A2 expression on T2 cells (FIG. 1, upperpanel). However, the NLMNLGATL (SEQ ID NO:6) analogue peptide stabilizedthe HLA-A2 molecule by showing a dose-dependent increase in HLA-A2expression, compared to the T2 cells without peptide pulsing (FIG. 1middle panel). Similar to the native peptide NQMNLGATL, NYMNLGATL (SEQID NO:7) peptide did not increase the HLA-A2 expression (FIG. 1 lowerpanel). These data confirmed the HLA-A2 biding scores, predicted by thecomputer-based algorithm.

Example 4 Induction of a Peptide-Specific of CD8 T Cell Response theContext of HLA-A0201 and A2402 Molecules

Although affinity for MHC molecules is necessary for the peptidepresentation, T cell recognition of the peptide presented by HLAmolecules is another important requirement for eliciting thepeptide-specific response. Therefore, using an in vitro stimulationprotocol, the new synthetic WT1 peptide analogs were evaluated for theirability to stimulate peptide-specific T cell response in both HLA-A0201and A2402 donors.

To expand the peptide-specific T cell precursors, three to five in vitrostimulation were performed and the specific T cell response was measuredby IFN-g production, when challenged with individual peptide. NLMNLGATLpeptide induced strong IFN-g secretion which crossed reacted with thenative NQMNLGATL peptide. Five stimulations of T cells enhanced theresponse showing by more IFN-g spots (FIG. 2B) than 3 stimulation (FIG.2A). T cells after 5 stimulation with NLMNLGATL peptide were also testedfor the cytotoxicity using ⁵¹Cr release assay. No killing was observedagainst HL-60 cells that were WT1 positive but HLA-A2 negative. However,the T cells killed the WT1+ and HLA-A0201+ AML cell line SET-2 andprimary leukemia blasts derived from a patient who is HLA-A0201 positive(FIG. 3). Whether both NLMNLGATL and NYMNLGATL heteroclitic peptidescould induce a better CD8 T cell responses in HLA-A2402 donors wasdetermined. NLMNLGATL peptide could induce T cell responses against bothNLMNLGATL and the native NQMNLGATL peptides, but there was nosignificant enhancement compared to the T cell response induced by thenative NQMNLGATL peptide. In the contrast, NYMNLGATL peptide induced astrong T cell response against itself and the native peptide after 3stimulation (FIG. 4A) but the response was demised after 5 roundstimulation (FIG. 4B), which also showed a weak cross reactivity withnative sequence. These data demonstrated that NLMNLGATL heterocliticpeptide is a strong epitope for CD8 T cells in the context of HLA-A0201molecule. NYMNLGATL peptide, on the other hand, induced CD8 T cellresponse in HLA-A0201 positive donors, but the response was notsignificantly better than the NQMNLGATL peptide.

Example 5 Induction of T Cell Response by HLA-DR.B1 Peptides thatRecognizes NQMNLGATL CD8 T Cell Epitope

It has been shown that a peptide combining both CD4 and CD8 epitopes ismore effective than the single class I epitope in eliciting effectiveimmune response for vaccine design, because CD4 T cells can help CD8 CTLby fully activating DCs through the CD40/CD40L signaling as well as byproducing IL-2 and IFN-g. In addition, if T cells stimulated with longerpeptides, in which CD8 T cell epitopes are imbedded in, could recognizethe short peptides, it would confirm the processing of the CD8 T cellepitopes. Therefore, four HLA-DR.B1-binding peptides that span theNQMNLGATL and NLMNLGATL epitopes, respectively, were designed:

(SEQ ID NO: 8) DR-Native-1: cmtwNQMNLGATLkg (SEQ ID NO: 9) DR-Native-2:wNQMNLGATLkgvaa (SEQ ID NO: 10) DR-het-1: cmtwNLMNLGATLkg(SEQ ID NO: 11) DR-het-2: wNLMNLGATLkgvaa

Since there is no definitive method to predict the class II peptidecleavage, two different versions of the class II peptides were designedusing the BIMAS, SYFPEITHI and RANKPEP algorithms (Table 2).

TABLE 2 Predictive binding scores of HLA-DRB binding peptides. SYFPEITHINative-1 DR.B1-0101 DR.B1-0301 DR.B1-0401 DR.B1-0701 DR.B1-1101DR.B1-1501 DR-Native 1 17  1 16 10 16  4 cmtwNQMNLGATLkg SEQ ID NO: 8Het-1 DR-het-1 18  2 16 10 16  4 cmtwNLMNLGATLkg SEQ ID NO: 10 Native-2DR-Native-2 17 13 14 16 13 24 wNQMNLGATLkgvaa SEQ ID NO: 9 Het-2DR-het-2 17 13 14 16 13 24 wNLMNLGATLkgvaa SEQ ID NO: 11

When T cells were stimulated with two “heteroclitic” DR.B1 peptidesspanning the NLMNLGATL epitope, they induced T cell responses that werespecific for both short and long peptides, showing by IFN-g secretion.Since CD4 peptides induce more potent response due to their massiveproduction of cytokines, the background is usually higher than CD8 Tcell peptide stimulation. Therefore, although both DR-heterocliticpeptides induced specific responses, DR-het-2 peptide showed a moreclear response than the DR-het-1 peptide in a donor shown in FIG. 5A. Itwas evident that DR-het-2 peptide induced responses were specific forboth short peptides NQMNGATL and NLMNGATL, and DR-native 2 and het-2peptides. More importantly, the responses were directed againstirradiated tumor cell line BA-25 (WT1+A2+), but not for the HL-60 cellsthat were WT1+ but A0201 negative. Similarly, when T cells werestimulated with short peptides (NQMNLGATL or NLMNGATL) or long peptidesas indicated in FIG. 5B, only BA-25 but not HL-60 cells were killed.

Example 6 Other HLA-DR.B1 Binding Peptides that Recognize NQMNLGATL CD8T Cell Epitope

In addition to those DR peptides described above, additionalHLA-DR.B1-binding peptides that span the NQMNLGATL, NLMNLGATL andNLMNLGATL epitopes were designed and evaluated (Table 3):

TABLE 3 Predictive binding scores of the peptides to HLA-DR.B1 SYFPEITHINative DR.B1-0101 DR.B1-0301 DR.B1-0401 DR.B1-0701 DR.B1-1101 DR.B1-1501cmtwNQMNLGATLkg 17  1 16 10 16  4 (SEQ ID NO: 8) mtwNQMNLGATLkgv 17 11 6  8  0  8 (SEQ ID NO: 12) twNQMNLGATLkgva 18  2 12  0  7  8(SEQ ID NO: 13) wNQMNLGATLkgvaa 17 13 14 16 13 24 (SEQ ID NO: 9) Het24-1cmtwNLMNLGATLkg 18  2 16 10 16  4 (SEQ ID NO: 14) mtwNLMNLGATLkgv 17 13 6  8  0  8 (SEQ ID NO: 15) twNLMNLGATLkgva 26 12 20  8 13 18(SEQ ID NO: 16) wNLMNLGATLkgvaa 17 13 14 16 13 24 (SEQ ID NO: 17)Het24-2 cmtwNYMNLGATLkg 17  1 16 10 16  4 (SEQ ID NO: 10)mtwNYMNLGATLkgv 17 11  6  8  0  8 (SEQ ID NO: 19) twNYMNLGATLkgva 28  222 10 17  8 (SEQ ID NO: 20) wNYMNLGATLkgvaa 17 13 14 16 13 24(SEQ ID NO: 11) RANKPEP DR.B1-0101 DR.B1-0301 DR.B1-0401 DR.B1-0701DR.B1-1101 DR.B1-1501 cmtwNQMNLGATLkgva 10.188; 3.577; 13.521;  7.85;21.138;  1.731; Native 21.12% 8.78% 30.67% 15.27% 32.2%  4.14%(SEQ ID NO: 21) Binder: Binder: Binder: wNQMNLGAT twNQMNLGA mtwNQMNLG(CMT-LKG- (CM-TLK- (C-ATL- 15 aa) 14 aa) 13 aa) (SEQ ID SEQ ID (SEQ IDNO: 24) NO: 26) NO: 28) cmtwNLMNLGATLkgva 9.377;  2.728; 11.145  7.85;22.089;  6.209; Het24-1 19.44% 6.7% 25.28% 15.27% 33.65% 14.84%(SEQ ID NO: 22) Binder: Binder: Binder: wNLMNLGAT MNLGATLkg mtwNQMNLG(CMT-LKG- (WNL-VA- (C-ATL- 15 aa) 14aa) 13 aa) SEQ ID (SEQ ID (SEQ IDNO: 25) NO: 27) NO: 28) cmtwNYMNLGATLkgva 7.184; 4.061; 11.145;  7.85;18.539;  8.439; Het24-2 14.89% 9.97% 25.28% 15.27% 28.23; 20.17%(SEQ ID NO: 23) Binder: Binder: MNLGATLkg mtwNQMNLG (WNY-VA- (C-ATL-14 aa) 13 aa) (SEQ ID (SEQ ID NO: 18) NO: 28)

Example 7 Generation of Peptides Derived from WT1 Oncoprotein that Bindto Human HLA-B7 Class I and HLA-Dr Class II Molecules

Peptides were also designed that that bind to HLA-B0702 (Table 4). Thefollowing peptide sequences were designed: RQRPHPGAL (B7-Native 1; SEQID NO:34), RLRPHPGAL (B7-het-1; SEQ ID NO:37), RIRPHPGAL (B7-het-2; SEQID NO:38), GALRNPTAC (Native 2; SEQ ID NO:29), and GALRNPTAL (B7-het-3;SEQ ID NO:31). The predictive binding scores of these and other variantsare shown in Table 4. These peptides were tested in vitro and stimulateheteroclitic T cell responses (FIG. 6). CD3 T cells from aHLA-B0702-positive donor were stimulated with 2 sets of peptides (totalfive) for 5 times in vitro. The peptide-specific response was measuredby IFN-gamma ELISPOT assay, against individual peptide.

For the first set of peptides, both heteroclitic-1 and 2, induced thepeptide-specific responses, but the cross reactivity to the native 1(N1) peptide was stronger for the het-2 than the het-1 peptide. For thesecond set of the peptides, heteroclitic peptide induced strong IFN-gproduction, when challenged with the stimulating peptide, but nocross-reactivity to the native sequence was found.

TABLE 4Predictive binding scores of B7 peptides to HLA-B7 and other haplotypes.RANKPEP B0702 SYFPEITH SYFPEITH SYFPEITH SYFPEITH SYFPEITH SYFPEITHSYFPEITH Score; % Opt I-B0702 I-A0201 I-A0301 I-A0101 I-B-08 B-2705I-B-3902 1. GALRNPTAC −18.084 −44.75%  2 14 (p −118 to −110) (B5101)SEQ ID NO: 29 GYLRNPTAC −20.632 −51.06% All SEQ ID NO: 30 below  8GALRNPTAL −9.401 −23.27% 12 18  8 16 17 20 SEQ ID NO: 31 (B5101)YALRNPTAC −14.528 −35.95% 10 All SEQ ID NO: 32 below 10 GLLRNPTAC −20.18−49.94%  2 14 18 14 SEQ ID NO: 33 2. RQRRHPGAL −3.687 −9.12% 15 13 13 1714 23 (p −125 to −117) (1501) SEQ ID NO: 34 RYRPHPGAL −4.517 −11.18% 1513 13 17 SEQ ID NO: 35 YQRPHPGAL −5.618 −13.90% 15 SEQ ID NO: 36RLRPHPGAL −4.065 −10.06% 15 23 23 23 17 SEQ ID NO: 37 (B37) RIRPHPGAL−2.674 −6.62% 15 21 21 21 SEQ ID NO: 38 BIMAS-B7 GALRNPTAC 0.3SEQ ID NO: 30 GALRNPTAL 12 SEQ ID NO: 31 RQRPHPGAL 40 SEQ ID NO: 34RLRPHPGAL 40 SEQ ID NO: 37 RIRPHPGAL 40 SEQ ID NO: 38

Based in the finding that the native peptides RQRPHPGAL (p-125 to -117;SEQ ID NO:34) and GALRNPTAC (p-118 to -110; SEQ ID NO:29) induce T cellsresponses in the context of HLA-B7 molecule, using HLA-bindingprediction algorithms, one heteroclitic peptide for the GALRNPTACpeptide was designed (SEQ ID NO:31), and two heteroclitic peptides forRQRPHPGAL (SEQ ID NOS:37 and 38). Based on the binding prediction, thesepeptides may also be able to stimulate T cells in the context of otherHLA haplotypes, such as: A0201, A0301, B8, B1501, B37 and B5101 (Table4).

Example 8 Generation of Peptides Derived from WT1 Oncoprotein that Bindto Human HLA-B35, A0101, A0301, A1101 Class I and HLA-DR Class IIMolecules

Peptide QFPNHSFKHEDPMGQ (p170-182) (SEQ ID NO:39) induces T cellsresponse in the context of HLA-DR.B1 0301 and 0401. The short sequencesimbedded within the long peptide, HSFKHEDPM, induces T cell response inthe context of B3501. Based on the predictions by the HLA-bindingprediction algorithms, one heteroclitic long peptide was designed, whichis the extension of the het-B35-1 short peptide.

The sequences of the peptides are: Class II peptide: DR.B1-03/04-Native:QFPNHSFKHEDPM (SEQ ID NO:42), DR.B1-03/04-Het: QFPNHSFKHEDPY (SEQ IDNO:43; Class I peptides: 1. Native: HSFKHEDPM (SEQ ID NO:40), 2.Het-01/03-1: HSFKHEDPY (for A0101 and A0301) (SEQ ID NO:41), and 3.Het-03/11-1 HSFKHEDPK (for A0301 and A1101) (SEQ ID NO:42). Heterocliticpeptides for the HLA-B3501 haplotype were tested in silico (Table 5).

TABLE 5Predictive binding scores of the natural peptides to HLA-DR.B1-0301, 0402 andB3501, A0101, A0301 and A1101. Class II SYFPEITHI (15 mer) DR.B1-0101DR.B1-0301 DR.B1-0401 DR.B1-0701 DR.B1-1101 DR.B1-1501 QFPNHSFKHEDPMGQ 82 12 0 14 14 SEQ ID NO: 39 RANKPEP QFPNHSFKHEDPMGQ −1.949; −2.786;6.717; 15.23% −4.04; 3.393; 8.864; SEQ ID NO:39 −4.04% −6.84% (0401)−8.56% 5.17% 21.18% 6.165; 13.82% (0402) Class I SYFPEITHI BIMAS RANKPEPHSFKHEDPM (B35-native) SEQ ID NO: 40 B3501 N/A 10 −3.568; −8.95% A0101 4 0.002 −16.2; −26.61% A0301  0 0.005 −3.832; −10.86% A1101 11 0−12.047; −30.69% HSFKHEDPY (B35-het1) SEQ ID NO: 41 B3501 N/A 10 −2.86;−7.18% Cleaved A0101 19 0.075 −5.164; −8.48% Cleaved A0301  6 0.1 7.127;20.20% Cleaved A1101 11 0 0.433; 1.1% Cleaved HSFKHEDPK (B35-het-2)SEQ ID NO: 42 B3501 N/A 0.05  −11.223;  −28.16% A0101  4 0.03  −15.83;−26% A0301 10 0.5    −8.783;   24.77% A1101 21 0.04     4.316;  11%

Example 9 Generation of Peptides Derived from WT1 Oncoprotein that Bindto Human HLA-A1, A3, all Class I and HLA-DR.B1-0401 Class II Molecules

Peptide KRPFMCAYPGCNK (320-332) (SEQ ID NO:44) was shown to induce Tcell response in the context of HLA-DR.B1 0401. The short sequenceimbedded within the long peptide, FMCAYPGCN (SEQ ID NO:45), induces Tcell response in the context of B35, B7 and A0101 (Table 6). The bindingscores were investigated of the peptides to multiple HLA haplotypesusing prediction algorithms. One heteroclitic long peptide was designed,which is the extension of the het-1 short peptide. Two shortheteroclitic peptides were designed that bind better to HLA-A0101, 0301and 1101. The sequences of the peptides are: Class II peptide: DR.B1-04Native: KRPFMCAYPGCNK (SEQ ID NO:44), DR.B1-04 het: KRPFMCAYPGCYK (SEQID NO:46); Class I peptides: 1. Native: FMCAYPGCN (SEQ ID NO:45), 2.DR.B1-04-Het-1 short: FMCAYPGCY (for A0101) (SEQ ID NO:47), 3.DR.B1-04-Het-2-short: FMCAYPGCK (for A0301 and A1101) (SEQ ID NO:48).KRPFMCAYPGCYK (SEQ ID NO:46) is the extension of DR.B1-04-het 1 short,FMCAYPGCN (SEQ ID NO:45), in which the end of the sequences CN becomesCY.

TABLE 6 Predictive binding scores of the peptides to HLA-DR.B1-0401 andB35, B7, A0101, A0301 and A1101. HLA-DR.B1 SYFPEITHI (15 mer) DR.B1-01010301 0401 0701 1101 1501 KRPFMCAYPGCNKRY 16 8 22 16 10 16 SEQ ID NO: 49KRPFMCAYPGCYKRY 16 16 16 22 10 12 SEQ ID NO: 55 SEKRPFMCAYPGCNK 15 0 0 012 8 SEQ ID NO: 50 RANKPEP KRPFMCAYPGCNK 5.381; −9.13; 3.131; −0.486;0.756; 4.199; SEQ ID NO: 44 11.15% −22.35% 7.1% −0.95% 1.15% 10.04%Class I SYFPEITHI BIMAS RANKPEP FMCAYPGCN (native) SEQ ID NO: 45 A0101 0 0.005 −4.165; −6.84% B7  1 0.02 −21.654; −53.59% B35 N/A N/A −23.926;−60.03% A0301  4 0.018 −2.503; −7.09% A1101  8 0 −2.509; −5.25%FMCAYPGCY (DR.B1-04-het1-short) SEQ ID NO: 47 A0101 15 0.25 6.613;10.86% Cleaved B7  1 0.02 −13.887; 34.37%, cleaved B35 N/A 0.02 −11.078;27.8% A0301 10 3.6 7.557; 21.42%, cleaved A1101  8 0.004 9.516; 24.24%,cleaved FMCAYPGCK (DR.B1-04-Het-2 short) SEQ ID NO: 48 A0101  0 0.14.053; −6.66% B7  1 0.01 −20.85, 51.71% B35 N/A 0.01 −21.886; 47.27%A0301 14 18 9.168; 25.99% A1101 18 0.4 8.883; 22.09%

Example 10 Additional Cross-Reactivity Studies

An ELISPOT assay was conducted using donor SA after 5 stimulations forthe Het24-1 (SEQ ID NO:6) and Het24-2 (SEQ ID NO:7) A24 peptides, incomparison to the native sequence (SEQ ID NO:5). As shown in FIG. 7, theheteroclitic peptides generate cross-reactive responses.

What is claimed is:
 1. An isolated peptide consisting of the amino acidsequence SEQ ID NO:6.
 2. An isolated peptide consisting of the aminoacid sequence selected from among SEQ ID NO:8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39,41, 42, 43, 44, 46, 47, 48, 49, 50 and
 55. 3. An isolated peptidecomprising the amino acid sequence SEQ ID NO:6
 4. An isolated peptidecomprising the amino acid sequence selected from among SEQ ID NO:8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33,35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and
 55. 5. Theisolated peptide of any one of claims 1-4, wherein said isolated peptidebinds to an HLA class I molecule, an HLA class II molecule, or thecombination thereof.
 6. An isolated class I binding peptide having SEQID NO:6.
 7. An isolated class I binding peptide selected from SEQ IDNO:30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and
 48. 8. An isolatedclass II binding peptide selected from SEQ ID NO: 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55.9. A pharmaceutical composition comprising a peptide of any one ofclaims 1-8 and a pharmaceutically acceptable carrier, vehicle orexcipient.
 10. A vaccine comprising (a) one or more isolated peptides ofclaims 1-8 or a pharmaceutical composition of claim 9, and (b) anadjuvant or a carrier.
 11. The vaccine of claim 10, wherein saidadjuvant is QS21, Freund's incomplete adjuvant, aluminum phosphate,aluminum hydroxide, BCG, alum, a growth factor, a cytokine, a chemokine,an interleukin, Montanide ISA 51, or GM-CSF.
 12. A method of treating asubject with a WT1-expressing cancer or reducing an incidence of aWT1-expressing cancer, or its relapse, the method comprisingadministering to said subject the vaccine of claim 10, thereby treatinga subject with a WT1-expressing cancer, reducing an incidence of aWT1-expressing cancer or its relapse therein.
 13. The method of claim12, wherein said WT1-expressing cancer is a leukemia, a desmoplasticsmall round cell tumor, a gastric cancer, a colon cancer, a lung cancer,a breast cancer, a germ cell tumor, an ovarian cancer, a uterine cancer,a thyroid cancer, a liver cancer, a renal cancer, a Kaposi's sarcoma, asarcoma, a hepatocellular carcinoma, a Wilms' tumor, an acutemyelogenous leukemia (AML), a myelodysplastic syndrome (MDS), or anon-small cell lung cancer (NSCLC).
 14. A method of inducing theformation and proliferation of CTL specific for cells of aWT1-expressing cancer, the method comprising administering to saidsubject the vaccine of claim 10, thereby inducing the formation andproliferation of CTL specific for cells of a WT1-expressing cancer. 15.The method of claim 14, wherein said WT1-expressing cancer is aleukemia, a desmoplastic small round cell tumor, a gastric cancer, acolon cancer, a lung cancer, a breast cancer, a germ cell tumor, anovarian cancer, a uterine cancer, a thyroid cancer, a liver cancer, arenal cancer, a Kaposi's sarcoma, a sarcoma, a hepatocellular carcinoma,a Wilms' tumor, an acute myelogenous leukemia (AML), a myelodysplasticsyndrome (MDS), or a non-small cell lung cancer (NSCLC).
 16. Acomposition comprising (a) an antigen-presenting cell and (b) a peptideof any one of claims 1-8.
 17. The method of any one of claims 12-16wherein the cancer is mesothelioma.